Targeted transposition for use in epigenetic studies

ABSTRACT

Disclosed herein are compositions, methods and kits useful for epigenetic analysis based on the use of transposons that are targeted to specific regions of chromatin based on DNA-DNA interactions, protein-protein interactions, RNA-RNA interactions, and nucleic acid-protein interactions.

FIELD OF THE INVENTION

The present invention is in the field of epigenetics. More specifically,compositions, methods and kits useful for epigenetic analysis based onthe use of transposons to specifically target specific regions ofchromatin.

BACKGROUND OF THE DISCLOSURE Overview of Epigenetic Mechanisms

Epigenetics is broadly defined as changes in phenotype that areheritable but do not involve changes in the DNA sequence, and, from ahistorical perspective, stems from long-standing studies of seeminglyanomalous (i.e., non-Mendelian) and disparate patterns of inheritance inmany organisms [1]. Examples include variation of embryonic growth,mosaic skin coloring, random X inactivation, and plant paramutation.Discoveries in a large number of different model systems have beenpivotal in identifying the three principle epigenetic mechanisms of (i)histone modifications, (ii) DNA methylation, and (iii) non-coding RNAs,which function in concert to influence cellular processes such as genetranscription, DNA repair, imprinting, aging, and chromatin structure,as depicted in FIG. 2.

Gene transcription occurs in the context of the nucleosomal structure ofchromatin. A nucleosome consists of an octamer of histone proteins (twomolecules of each core histone H2A, H2B, H3, and H4) around which iswrapped 147 base pairs (bp) of DNA. Histones are small basic proteinswith an unstructured amino-terminal “tail” that are the target ofnumerous post-translational modifications [2, 3]. Specific histone marksin the fission yeast Saccheromyces pombe were demonstrated to bedirectly operating as activating and repressing signals for genetranscription [4]. Methylation of lysine 4 and acetylation of lysine 9of histone H3 are associated with transcriptionally active chromatin,while methylation of lysine 20 of histone H4 and methylation of lysine 9and 27 of histone H3 are repressive marks, found in transcriptionallysilent heterochromatin regions [5, 6]. The repressive histone H3 lysine9 trimethyl-mark is bound by HP1 proteins, which in turn recruitnon-coding RNAs involved in regulating heterochromatin formation [7].

Similar mechanistic links have also been identified between histonemarks and DNA methylation. Highly repetitive DNA tandem repeat sequencessuch as those found in pericentric heterochromatin rely on therepressive H3K9 methylation mark to direct de novo DNA methylation whileat promoters, EZH2, a histone lysine methyltransferase containingcomplex is involved [8]. Members of the methyl-CpG binding domain (MBD)family of proteins which are readers of DNA methylation are found incomplexes with histone modifying enzymes (MeCP2 recruits histonedeacetylases to mediate histone repressive marks [9]). Studies inmulticellular organisms such as the invertebrates Caenorhabditis elegansand Drosophila melanogaster and plants such as Arabidopsis thaliana havegenerated crucial links between these epigenetic mechanisms [10].

In spite of all the advances to date, however, the epigenetics researchfield is still in the discovery phase, with many mechanistic questionsremaining unanswered and many key players yet to be identified. Just asin the past, the continued study of epigenetic mechanisms in a varietyof model organisms will be required to answer these questions.Development of enabling technologies suitable for a broad spectrum ofmodel systems are also critical for accelerating the rate of discovery,especially since the various epigenetic mechanisms are functionallyinterconnected.

Chromatin Immunoprecipitation (ChIP)

ChIP was first described in 1993 following studies of the association ofhistone acetylation state with transcriptional gene silencing in yeast[11]. Its adaptation to mammalian cells was reported five years later,in 1998 [12]. Since its initial description, the technique has remainedessentially unchanged. As described below and depicted in FIG. 1, PanelA, DNA sequence analysis is performed on the fraction of DNA isolated byimmunoprecipitation with antibodies specific to the protein of interest.This technique is used in a wide variety of applications. These includeprofiling histone modification patterns, from their intragenicdistribution through to genome-wide analysis, determining thecomposition of protein complexes recruited by specific histone marks,identifying regions of de novo DNA methylation, or, with somemodifications to the procedure, detecting nascent non-coding RNAs.

Advances in PCR and DNA sequencing technologies have positively impactedthe DNA analysis portion of the ChIP technique, which has expanded fromsemi-quantitative analysis of single genes using end-point PCR, toquantitative analysis with real-time PCR, through to genome-wideanalysis afforded by ChIP-ChIP, wherein the captured DNA is used toprobe a high-density microarray, or ChIP-Seq, wherein the captured DNAis subjected to NGS (“next generation sequencing”) [6, 13]. While theseimprovements have increased the magnitude of sequence informationavailable for analysis from a single reaction, the limitationsassociated with efficient immunocapture of protein-associated DNA havenot been addressed.

Only incremental improvements, such as the introduction of magneticbeads for immunocapture in place of agarose or sepharose beads, as inActive Motifs ChIP-IT Express™ kit, have been made [14]. The improvedrecovery (fewer beads are lost during wash steps), reduced background(wash steps are more thorough) afforded through the use of magneticbeads has allowed for a ten-fold reduction in the sample sizerequirements, from 2-10 million cells to 0.1-1 million cells. Ingeneral, these lower sample requirements apply only to high affinityantibodies targeting abundant proteins, such as RNA polymerase II orhistone modifications. In addition, the sample size requirement remainsa considerable barrier in some research areas, such as embryology andstem cells where cell numbers are very limiting, and is furthercompounded by the limitation that the only a single protein can beanalyzed in each ChIP experiment. The number of cells required is thusdirectly proportional to the number of proteins to be analyzed,impacting cost and time considerations. An additional challenge stemsfrom the need of ultra-high affinity antibodies for use in thistechnique. Many antibodies qualified for use in immunofluorescenceand/or immunohistochemistry, which can be used to demonstrate in situassociation of the protein of interest with DNA or chromatin, orantibodies which have been shown to effectively function inimmunoprecipitation, fail in ChIP applications where the target proteinis present in high molecular weight multi-protein-chromatin complexescontaining DNA fragments up to 1 kb (kilobase) in length. The bindingaffinity of the antibody for its cognate target must be strong enough towithstand the physical forces associated with constant agitation of thesuspension and immobilization by the beads used to isolate thecomplexes.

Chirp (Chromatin Isolation by RNA Purification) and CHART (CaptureHybridization Analysis of RNA Targets)

Non-coding RNAs (ncRNAS) have multiple functions in the cell, forexample, one described function is for the RNA molecule itself tofunction as a scaffold that directs and maintains the assembly andstability of multiprotein complexes. These complexes often containchromatin targeting and chromatin modifying proteins that assemble intoDNA as part of the overall chromatin structure.

Since ncRNAs are known to be part of important chromatin modifyingcomplexes, techniques have been developed to identify how such RNAinteracts with DNA across the genome, for example, Chirp and CHART. BothChirp and CHART are essentially the same and are described in briefbelow.

-   -   1. A Series of biotinylated oligonucleotides are designed to        hybridize to the ncRNA of interest.    -   2. Formaldehyde fixed chromatin is prepared (similar to what is        done for ChIP)    -   3. Oligonucleotides are hybridized to the native target within        the chromatin prep    -   4. Streptavidin beads are used to pull out the ncRNA of        interest, associated proteins and associated genomic DNA    -   5. After isolation and clean-up of genomic DNA, libraries are        prepared and the DNA is sequenced using Next-Gen sequencing        platforms such as Illumina.        These techniques are very similar to ChIP but rather than using        an antibody to isolate the genomic DNA associated with proteins,        oligonucleotides are used to isolate the genomic DNA associated        with ncRNAs [38-40].

Need for and Benefits of the Invention

The instant invention has broad and significant practical applications.These applications span all life sciences research with eukaryoticorganisms, because epigenetic mechanisms are highly conserved throughouteukaryotes. The methods of this invention are more efficient thanexisting methods such as ChIP. These new, patentable methods enableconcurrent analysis of multiple chromatin-associated proteins, eliminatethe labor intensive NGS library preparation procedures, and have thepotential to significantly reduce the amount of samples needed comparedto traditional ChIP methods. This is relevant to not only to the stemcell and embryology research fields where samples are limiting, but alsofields such as high throughput screening of large numbers of samples inclinical and pharmaceutical applications, where miniaturization is amajor cost driver. In addition, ChIP analysis is limited by the smallpercentage of antibodies that work effectively in the method. Since themethods of the invention do not require immunoprecipitation, antibodiesthat do not work in ChIP can be adapted to work with the instantinvention, thereby expanding the number of cellular proteins whosegenomic distribution can now be determined.

SUMMARY OF THE INVENTION

One aspect of the invention concerns methods and reagents for making anucleic acid sequence library or libraries. Such methods involveextracting and optionally fragmenting chromatin from a prepared sample,adding at least one protein-oligonucleotide conjugate comprising anextraction moiety, allowing said protein(s) to locate at its/theirtarget protein(s) and or DNA-binding sites, and or RNA-binding sties insaid chromatin fragments, tagging the nucleic acid in said chromatinfragments with said conjugate by inducing an intermolecular reactionbetween said oligonucleotide and said nucleic acid, extracting thenucleic acid so tagged using the extraction moiety. In other aspects,the extracted tagged nucleic acid is sequenced.

Another aspect of the invention concerns methods and reagents for makinga nucleic acid sequence library or libraries. Such methods involveextracting and optionally fragmenting chromatin from a prepared sample,adding at least one oligonucleotide-transposome construct comprising anextraction moiety, allowing said oligonucleotide-transposome constructto locate at its/their DNA and/or noncoding RNA-binding sites in saidchromatin, tagging the nucleic acid in said chromatin fragments withsaid construct by inducing an intermolecular reaction between saidoligonucleotide and said nucleic acid, extracting the nucleic acid sotagged using the extraction moiety. In a related embodiment theoligonucleotide contains peptide nucleic acid that targets G-quadruplexstructures. In other aspects, the extracted tagged nucleic acid issequenced.

The methods disclosed herein can be applied to eukaryotic andprokaryotic, e.g., bacterial organisms [43-46].

The methods disclosed herein can be applied to samples in which thechromatin has been crosslinked to proteins in vivo or samples withoutcrosslinking.

In some embodiments, the protein-oligonucleotide conjugate oroligonucleotide-transposome construct further comprises transposase andthe intermolecular reaction is transposition, the extraction moiety is abiotin molecule, and/or the intermolecular reaction is selected from thegroup: transposition, ligation, recombination, hybridization, andtopoisomerase-assisted insertion.

A related aspect of the invention concerns antibody-transposomecomplexes. Such complexes comprise an antibody that binds a targetnucleic acid-associated protein conjugated to a transposome thatcomprises a transposase and a transposon cassette.

In still another related aspect, disclosed herein are protein-tansposomecomplexes. Such complexes comprise a protein that binds, withoutlimitation, a protein-binding partner, methylated DNA, non-coding RNA,and/or DNA-binding site. In another embodiment, the protein is anantibody or antibody fragment (both encompassed by the term antibody).In still another embodiment, the protein contains particular bindingmotifs, such as, without limitation, bZIP domain, DNA-binding domain,helix-loop-helix, helix-turn-helix, MG-box, leucine zipper, lexitropsin,nucleic acid simulations, zinc finger, histone methylases, recruitmentproteins, Swi6, chromodomain, chromoshadow domains, bromodomains, orPHD-finger. In some embodiments, the protein is MBD2 or MBD3.

In still another related aspect, disclosed herein areoligonucleotide-transposome constructs. Such constructs comprise anoligonucleotide that targets non-coding RNA and/or G-quadruplexstructures. In a related embodiment, the oligonucleotide can containlocked nucleic acids and/or peptide nucleic acid-nucleic acid chimeras.

In some embodiments, the transposome is comprised of Tn5 or TS-Tn5transposon.

An embodiment disclosed herein are kits including reagents,protein-transposome complex(s) and/or oligonucleotide-transposomeconstruct(s), and instructions for their use.

Another aspect of the invention relates to methods for performingproximity ligation. Such methods include contacting a crosslinked andfragmented chromatin sample with an antibody-oligonucleotide conjugateunder dilute conditions to promote ligation of the ends of the chromatinfragment to the ends of the oligonucleotide of theantibody-oligonucleotide conjugate, wherein the oligonucleotide isdouble stranded and comprises at least two recognition sites for afreeing restriction enzyme, primer sites for amplification, at least onebar code sequence to identify the conjugated antibody, complementaryoverhangs to facilitate ligation, and optionally, a spacer foroptimizing the length of the oligonucleotide, and then ligating theantibody-oligonucleotide conjugates to the crosslinked and fragmentedchromatin sample.

A related aspect involves antibody-oligonucleotide conjugates useful forproximity ligation reactions. These typically comprise an antibody thatbinds a target nucleic acid-associated protein that is conjugated to adouble-stranded oligonucleotide that comprises at least two recognitionsites for a freeing restriction enzyme, primer sites for amplification,at least one bar code sequence to identify the conjugated antibody,complementary overhangs to facilitate ligation, and optionally, a spacerfor optimizing the length of the oligonucleotide.

Another embodiment disclosed herein are methods to enrich for DNAmethylated genomic regions using transpososome-antibody/Oligonucleotidecomplex as described in Example 14 and FIG. 40. In an aspect of thisembodiment, the enrichment is performed using unfragmented genomic DNAor Chromatin.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—shows a comparison of ChIP-Seq Methods

FIG. 2—shows some epigenetic mechanisms that interact to influence genetranscription.

FIG. 3—shows a schematic diagram of Tn5 “cut and paste” transposition

FIG. 4—shows an evaluation of Nextera Tagmentation in ChIP-Seq DNALibrary Preparation. Q-PCR was used to detect enrichment of p53 bindingsites in p53 immuno-enriched chromatin from stimulated MCF7 human breastcancer cells.

FIG. 5—shows a schematic of experiments following identification ofEZ-Tn5 compatible ChIP cell lysis buffer

FIG. 6—shows a schematic of conjugation scenarios

FIG. 7—experiment sequence for optimal TA-ChIP methodology development

FIG. 8—shows one approach for proximity ligation

FIG. 9—shows a Schematic representation of Illumina-compatiblesequencing library preparation generated by the Nextera Kit.Transposomes assembled with free ends appended with sequencing tags (a)are used in a tagmentation reaction to produce a 5′-tagged DNA fragmentlibrary. Limited-cycle PCR with a four-primer reaction (4pPCR) addsIllumina bridge PCR—compatible adaptor sequences (b). Optionalbarcoding/indexing (triangle) is incorporated into Adapter 2 (andoptionally also Adapter 1) and added between the upstream bridge PCRadaptor and sequencing tag 2 (c). Features of the amplicons producedwith comprise the NGS library. (Adapted from www.Epicentre.com)

FIG. 10—shows Tn5 Transposase activity on Hela DNA and chromatin.Illumina Tagment DNA buffer and Tn5 (A) or TS-Tn5 (B) transposase,ranging from 5 to 20 Units, were added to HeLa DNA. The samples wereincubated at 55° C. for 5 minutes and allowed to cool to 10° C. Onefourth of the tagmented DNA was analyzed on a 1.5% agarose gel. C−, DNAwith no transposase added. C. Tagmentation of Hela chromatin DNA. 25Units of Tn5 transposome was added to 10 μg of crosslinked HeLachromatin sonicated to varying extents. Tagmented chromatin was treatedwith RnaseA and Protein K and crosslinks reversed followed bypurification. For the lane marked 20F, tagmentation products were firstpassed through a size exclusion spin column to remove low molecularweight DNA fragments and primers as well as salts and other impuritiesthat could impede with the downstream PCR reaction. 100 ng of tagmentedDNA was subjected to 15 cycles of PCR and one fifth of the PCR reactionvolume was analyzed on a 1.5% agarose gel. gDNA=Naked genomic DNAsubjected to tagmentation, purification and 100 ng of the purifiedproduct subjected to subsequent PCR amplification. MW markers: lowestband in M100=100 bp; Lowest band on M1 kb=0.5 kB. next band=1. Arrowdenotes position of input, untagemented DNA. Bracket denotes migrationsmear of randomly tagmented DNA fragments of varying sizes.

FIG. 11—shows the secondary and primary conjugation scheme.

FIG. 12—shows Analysis of secondary antibody conjugate on 10% nativepolyacrylamide gel A. 2 μg of conjugated antibody loaded on a 10% nativepolyacrylamide gel and stained with GelRed nucleic acid stain. 1indicates antibody—oligonucleotides conjugate zone and 2oligonucleotides zone B. 1/150 of isolated chromatography peaks 1 and 2from FIG. 16 loaded on a 10% native polyacrylamide gel and stained withGelRed nucleic acid stain. 1 indicates Chromatography peak “1” and 2Chromatography peak “2”. Peak “1” consists of pureantibody-oligonucleotide conjugate and is devoid of freeoligonucleotide.

FIG. 13—shows a Comparison of fragmentation of genomic DNA by Tn5 andTS-Tn5 transposome-antibody conjugates at different temperatures andfree Tn5 enzyme under standard conditions. 25 units of Tn5 or TS-Tn5transposase assembled with antibody-oligonucleotide conjugate were addedto 1 ug of genomic MCF7 DNA in Tagment DNA buffer. The samples wereincubated at 55° C. for 1 hour (C+ lanes only) or 37° C. for 1 to 3hours. Half of the tagmented DNA was analyzed on a 1.5% agarose gel. C−,no transposase. Arrow denoted untagmented input DNA. Bracket denotestagmented DNA fragments.

FIG. 14—shows Transposome-secondary antibody complex is directly toH3K4me3 antibody binding sites. The H3K4me3 primary antibody wasincubated with 0.1 or bug of chromatin overnight. Either the Tn5 orTS-Tn5 transposome-antibody conjugate was added at of 1:1 of primaryantibody to secondary-transposome conjugate ratio), and incubated at 4degrees for four hours to allow binding of the secondaryantibody-transposome to the primary antibody. Samples were diluted fourvolumes of IP dilution buffer followed by one volume of tagmentationbuffer containing Mg²⁺ to activate the transposase and incubated at 37degrees for 3 hours. The chromatin bound and tagmented by thetransposomes was captured using Protein G agarose beads (Invitrogen) andeluted in buffer containing the reducing agent TCEP(Tris(2-carboxyethyl)phosphine hydrochloride) to sever the cleavabledisulfide bond used to generate antibody-oligonucleotide conjugate.Proteins were removed by digestion with the protease Proteinase K andformaldehyde cross-links reversed as per established ChIP protocols.Half of the eluted DNA was subjected to 25 cycles of PCR using theapproach illustrated in FIG. 2. PCR amplification products were purifiedusing standard methods and diluted to 2 ng/ul. Quantitative PCR wasperformed using primers targeting the regions known to be negative(Untr12 and Untr20) or positive (GAPDH and Zc3h13) for H3K4me3 and 10 ngof DNA.

FIG. 15—NGS profile of the TAM-ChIP library relative to a traditionalChIP library. Traditional ChIP and TAM-ChIP was performed on 10 μg ofMCF7 chromatin with 2 μg of H3K4me3 antibody. Libraries were preparedusing standard protocols for the traditional library and as described inFIG. 6 for TAM-ChIP. The enriched DNA was sequenced on the IlluminaMiSeq platform. Regions of enrichment from A, a 4 Mb region fromchromosome 21, and B, a 3.05 Mb region from chromosome 13 are shownabove.

FIG. 16—shows Tn5 Transposase activity on Hela DNA and chromatin.Illumina Tagment DNA buffer and Tn5 (A) or TS-Tn5 (B) transposase,ranging from 5 to 20 Units, were added to HeLa DNA. The samples wereincubated at 55° C. for 5 minutes and allowed to cool to 10° C. Onefourth of the tagmented DNA was analyzed on a 1.5% agarose gel. C−, DNAwith no transposase added. kb·C. Tagmentation of Hela chromatin DNA. 25Units of Tn5 transposome was added to 10 μg of crosslinked HeLachromatin sonicated to varying extents. Tagmented chromatin was treatedwith RnaseA and Protein K and crosslinks reversed followed bypurification. For the lane marked 20F, tagmentation products were firstpassed through a size exclusion spin column to remove low molecularweight DNA fragments and primers as well as salts and other impuritiesthat could impede with the downstream PCR reaction. 100 ng of tagmentedDNA was subjected to 15 cycles of PCR and one fifth of the PCR reactionvolume was analyzed on a 1.5% agarose gel. gDNA=Naked genomic DNAsubjected to tagmentation, purification and 100 ng of the purifiedproduct subjected to subsequent PCR amplification. MW markers: lowestband in M100=100 bp; Lowest band on Mlkb=0.5 kB. next band=1. Arrowdenotes position of input, untagemented DNA. Bracket denotes migrationsmear of randomly tagmented DNA fragments of varying sizes.

FIG. 17—Transposase efficiency on Lambda DNA and genomic HeLa DNA in 2fold dilutions of ChIP buffer. Varying dilutions of ChIP lysis buffercontaining either Lambda or genomic HeLa DNA were mixed with an equalvolume of 2× Tagment DNA buffer and the Tn5 enzyme (Nextera Kit).Samples were incubated at 55° C. for 5 minutes, then cooled to 10° C.Tagmented DNA was purified and eluted in 25 μl. One fifth of the elutedDNA was analyzed on a 1% agarose gel. Un, undiluted ChIP buffer; C, DNAwith no transposase added. M100, a 100 bp molecular weight DNA ladder.

FIG. 18—shows comparisons of Epicentre and Illumina oligonucleotidesequences. During the assembly of the transposome complex thetransposase enzyme will bind to a 19 bp double stranded transposon DNA(purple). Illumina complete changed the sequence of the single strandedappended ends seen in black to make the subsequent tagmented DNAcompatible with their index adapters and primers in the Nextera Indexkit. A-METS EPICENTRE is SEQ ID NO: 9; A-METS Illumina is SEQ ID NO: 10;B-METS EPICENTRE is SEQ ID NO: 11; B-METS Illlumina is SEQ ID NO: 12;p-MENTS is SEQ ID NO: 13

FIG. 19 (A). Activity of in house assembled Tn5 and TS-Tn5 Transposomeson genomic HeLa DNA. Illumina Tagment DNA buffer and Tn5 or TS-Tn5transposomes (A+B), ranging from 5 to 20 Units, were added to HeLa DNA.The samples were incubated at 55° C. for 5 minutes and then allowed tocool to 10° C. One fourth of the tagmented DNA was analyzed on a 1.5%agarose gel. C−, DNA with no transposase added. MW markers: lowest bandin M100=100 bp; Lowest band on Mlkb=0.5 kB., next band=1 kb (B)Comparison of fragmentation of genomic DNA by Tn5 and TS-Tn5transposome-antibody conjugates at different temperatures and free Tn5enzyme under standard conditions. 25 units of Tn5 or TS-Tn5 transposaseassembled with antibody-oligonucleotide conjugate were added to 1 ug ofgenomic MCF7 DNA in Tagment DNA buffer. The samples were incubated at55° C. for 1 hour (C+ lanes only) or 37° C. for 1 to 3 hours. Half ofthe tagmented DNA was analyzed on a 1.5% agarose gel. C−, notransposase. Arrow denoted untagmented input DNA. Bracket denotestagmented DNA fragments.

FIG. 20—Comparison of Illumina's and Applicant's tagmentation buffers.Activity of in house assembled Tn5 and TS-Tn5 transposomes on genomicHeLa DNA was determined using either a 5× Tagment buffer (lanes 1-4)made by Applicant with the 2× Illumina Tagment DNA buffer (lanes 5-8),and 5 units of Tn5 or TS-Tn5 transposomes assembled with either doublestranded A-METS only or B-METS only (indicated as A or B), to genomicHeLa DNA. The samples were incubated at 55° C. for 5 minutes and thencooled to 10° C. One fourth of the tagmented DNA was analyzed on a 1.5%agarose gel.

FIG. 21—Effect of varied oligonucleotide:Transposase ratios ontransposome activity. Varying amounts of Tn5 and TS-Tn5 transposase wereincubated with double stranded oligos at ratios ranging from 1:1 to1:100. Tagment DNA buffer was added with 5 units enzyme to genomic HeLaDNA and incubated at 55° C. for 5 minutes and then reactions cooled to10° C. One fourth of the tagmented DNA was analyzed on a 1.5% agarosegel. Arrows denote migration of free oligonucleotides.

FIG. 22 Compatibility of tagmented DNA produced with in housetransposomes with the Associated Nextera Index kit. Limited-cycle bridgePCR (5 cycles), using the Associated Nextera Index kit adapters andprimers, on 100 ng of DNA, tagmented by the in house generated Tn5 andTS-Tn5 transposomes. One fifth of the PCR amplification was analyzed ona 1.5% agarose gel. C+ indicates genomic HeLa DNA tagmented by the Tn5transposome. C− indicates no added transposome.

FIG. 23 shows Limited PCR of Tn5 tagmented genomic HeLa DNA withdifferent combinations of Illumina and Applicant's primers and adapters.One fifth of each PCR reaction was run on a 1.5% agarose gel. lxindicates 10 μM of Applicant's Primer 1+2 and 0.5 μM of each Adapter. 5×corresponds to 50 μM of Primer 1+2 and 2.5 μM of each Adapter. Lane 1and 2: Applicant Adapter 1a and 2a plus Primer 1 and 2, Table 1. Lane 3and 4: Applicant Adapter 1b and 2b plus Primer 1 and 2, Table 3. Lane 4(C+): Illumina's Nextera Primer Cocktail (Primer 1 and 2, FIG. 19) andIndex 1 and 2 adapters (Adapter 1 and 2, FIG. 19). Lane 5 (Ca): NexteraPrimer Cocktail and Applicant's Adapters 1 and 2a (lx). Lane 6 (Cb):Nextera Primer Cocktail+Applicant's Adapters 1 and 2b (lx). Lane 7(C1+2): Applicant's Primer 1+2 mix (lx) and Illumina's Index 1 and 2Adapters. Unincorporated primers and adapters are visualized as the bandat the bottom of the gel, increasing in intensity with increased amountsof primers and adapters.

FIG. 24—Tn5 Transposase tagmentation activity on crosslinked HeLachromatin. Tagment DNA buffer and Tn5 transposome, ranging from 50 to100 Units, were added to 10 μg of crosslinked HeLa chromatin. Thesamples were incubated at 55° C. for 5 then cooled to 10° C. Thetagmented chromatin was treated with RnaseA and Protein K and crosslinksreversed followed by DNA purification. 100 ng of tagmented DNA wassubjected to 25 cycles of PCR. A. Half of the PCR was analyzed on a 1.5%agarose gel. C+ indicates tagmented genomic HeLa DNA. B. The remaininghalf of the PCR sample from lane one was purified using Agencourt AMPureXP magnetic beads to remove un-integrated adapters and primers and 500ng of the PCR run on a 1.5% agarose gel.

FIG. 25—shows optimal ratio of primary versus secondary antibody inChIP. 10 μg of MCF7 crosslinked chromatin was used in ChIP forimmunoprecipitation with 2 ug of H3K4me3 antibody and varying amounts ofsecondary IgG. The antibody bound chromatin was captured with Protein Gagarose beads and subjected to Proteinase K treatment and reversal ofcrosslinks. 5% of the eluted IP was used for qPCR analysis with primersagainst Untr12 and GAPDH.

FIG. 26—shows Synthesis of nitro-SDPD. Mercaptopropionic acid inacetonitrile was treated with 2,2′-dithiobis(5-nitropyridine) in thepresence of triethylamine. Citric acid solution was added and theresulting 3-([5-nitro-2-pyridyl]dithio) propionic acid was extractedwith dichloromethane. The product was purified by silica gel flashchromatography. To prepare an active form, 3-([5-nitro-2-pyridyl]dithio)propionic acid and N-hydroxysuccinimide were dissolved in acetonitrileand N,N′-dicyclohexylcarbodiimide was added. Once the reaction wascomplete, crude nitro-SPDP was purified by preparative thin layerchromatography.

FIG. 27 shows Analysis of secondary antibody conjugate on 10% nativepolyacrylamide gel 2 μg of conjugated antibody loaded on a 10% nativepolyacrylamide gel and stained with GelRed nucleic acid stain. 1indicates antibody—oligonucleotides conjugate zone and 2oligonucleotides zone. A. 1/150 of isolated chromatography peaks 1 and 2from FIG. 16 loaded on a 10% native polyacrylamide gel and stained withGelRed nucleic acid stain. 1 indicates Chromatography peak “1” and 2Chromatography peak “2”. Peak “1” consists of pureantibody-oligonucleotide conjugate and is devoid of freeoligonucleotide.

FIG. 28—shows secondary antibody retains binding activity afterconjugation. 10 μg of MCF7 crosslinked chromatin was used in ChIP forimmunoprecipitation with 2 μg of H3K4me3 antibody and 4 ug (1) or 2 ug(2) of secondary antibody-conjugate and compared with 6 ug (3), 4 ug (4)2 ug (5) or 1 ug (6) of unconjugated secondary antibody and (7). Noprimary antibody and 2 μg of secondary antibody-conjugate.

FIG. 29—Tagmentation of genomic DNA by the Tn5 transposase assembled tooligonucleotide-antibody conjugate at different concentrations and timelengths. Tagment DNA buffer and varying amounts of the Tn5transposome-conjugate was incubated with 1 μg of genomic MCF7 DNA at 55°C. degrees for 10 and 30 minutes. Half of the tagmented DNA was analyzedon a 1.5% agarose gel. C− indicates DNA with no transposase added.

FIG. 30—Tagmentation of genomic DNA by Tn5 assembled tooligonucleotide-antibody conjugate at different temperatures. TagmentDNA buffer and 25 units of the Tn5 transposome-conjugate was incubatedwith 1 μg of genomic MCF7 DNA at either 37° C. or room temperature forvarious time lengths. Half of the tagmented DNA was analyzed on a 1.5%agarose gel. C+ indicates Tn5 transposome incubated at 55° C. for onehour.

FIG. 31—shows Tn5 and TS-Tn5 Transposome—Conjugate efficiency on genomicMCF7 DNA. Tagment DNA buffer and 25 units of Tn5 or TS-Tn5 transposaseassembled with antibody-oligonucleotide conjugate was added to 1 μg ofgenomic MCF7 DNA. The samples were incubated at 55° C. for one hour (C+)or 37° C. for one to three hours. Half of the tagmented DNA was analyzedon a 1.5% agarose gel. C−, DNA with no transposase added.

FIG. 32—shows Quantitative PCR analysis of Tn5 and TS-Tn5transposome/antibody conjugates in ChIP with H3K4me3 primary antibody.10 ug of cross-linked MCF7 was used in ChIP with 4 μg of H3K4me3antibody and 4 μg secondary antibody-conjugate. Tagmentation buffer wasadded after allowing the secondary antibody-transposome conjugate tobind the H3K4me3 and the bound chromatin captured with Protein G agarosebeads.

FIG. 33—QPCR profile without four primer PCR library amplification. Halfof the eluted ChIP DNA from the ChIP experiment in FIG. 31 was dilutedto mimic the bPCR step, purified with Agencourt AMPure beads and 10 ngsubjected to qPCR using primers against Untr12 and GAPDH.

FIG. 34—shows optimization of TTAM-ChIP. The numbers along the x-axisdirectly correspond in sequence to the following conditions listed inthe embedded figure legend.

FIG. 35—SeqMINER promoter profiles of the TAM-ChIP and traditional ChIPNGS HiSeq data. Tag densities from each ChIP-seq dataset were collectedwithin a window of 10 kb around the reference coordinates, the collecteddata were subjected to k-means clustering (using linear normalization).Using seqMINER, the average profile for selected clusters wasautomatically calculated and plotted. The H3K4me3 mean profile fortraditional ChIP-seq (graph with highest peak) and TAM-ChIP-seq (graphwith lowest peak) was calculated and represented.

FIG. 36—Detection of goat antibody using immunoPCR. Detection antibodyis oligonucleotide conjugated anti-goat IgG.

FIG. 37—shows the traditional MethylCollector™ approach that is modifiedto include the transposase.

FIG. 38—shows the modified MethylCollector™ protocol to determine thefeasibility of altering the current protocol to include the transposomerequired conditions.

FIG. 39—shows DNA methylation enrichment using the modifiedMethylCollector™ protocol

FIG. 40—shows the modified MethylCollector™ protocol to become TAM-MIRA.

FIG. 41—shows TAM-MIRA to determine 5-mC levels in human genomic DNA.

FIG. 42—shows TAM-MIRA to determine 5-hmC levels in mouse genomic DNA

DETAILED DESCRIPTION OF THE EMBODIMENTS

The disclosure herein provides methods of tagging and isolating DNA orother nucleic acids that are associated with a protein or proteins ofinterest. Generally the methods comprise first preparing complexes ofoligonucleotide tag(s) or barcode(s) with antibody(ies) that recognizeprotein(s) of interest in chromatin or that are otherwise associatedwith nucleic acids. The tagged oligonucleotide complexes may furthercomprise an extraction moiety, such as a biotin molecule (or othermember of a high affinity binding pair), that can be used to extract orisolate the tagged nucleic acid. A “binding partner” or “member” of ahigh affinity binding pair (i.e., a pair of molecules wherein one of themolecules binds to the second molecule with high affinity (e.g., biotinand avidin (or streptavidin), carbohydrates and lectins, effector andreceptor molecules, cofactors and enzymes, enzyme inhibitors andenzymes, and the like).

Next, when the complexes are added to the nucleic acids, theantibody(ies) recognize or bind to the protein(s) of interest that areassociated with the nucleic acids. Using a variety of intermolecularreactions, the nucleic acid proximate those proteins is tagged with thecomplex. Thus, the proximate nucleic acid is tagged with one or moreoligonucleotide bar code(s) and, optionally, a moiety that allows forpurification or isolation.

One embodiment of the invention, termed “Transposase-AssistedMulti-analyte Chromatin ImmunoPrecipitation” or “TAM-ChIP”, is a uniquemethod that significantly improves ChIP, the principle techniquecurrently used to study how histone post-translational modifications andthe proteins which they recruit regulate gene expression. TraditionalChIP is a cumbersome multiday, multistep procedure that requires largenumbers of cells, ultra-high affinity antibodies for the immunocaptureof large protein-chromatin complexes, and is limited to the analysis ofa single protein species per sample.

Briefly, conventional ChIP methods involve the crosslinking of DNA andprotein in live cells, isolation of crosslinked material, shearing ofDNA (still bound, through crosslinking, to protein), immunoprecipitationof the crosslinked DNA-protein complexes via antibody-binding of theprotein of interest (still bound to DNA), reverse-crosslinking of DNAand proteins, and the detection or sequencing of DNA molecules that werecrosslinked to the immunoprecipitated DNA-protein complexes, allowingthe generation of specific, DNA sequence context data (FIG. 1, Panel A).For ChIP-Seq applications, the elapsed time from formaldehydecrosslinking of cells to sequencing-ready library is typically fivedays. Relative to the advances made in the understanding the epigeneticmechanisms of DNA methylation and micro- or non-coding RNAs, thelimitations of the ChIP technique have significantly hampered theunderstanding of the biological function of histone modifications.

In contrast, TAM-ChIP (FIG. 1, Panel B) removes a number of technicaland sample-size barriers associated with traditional ChIP (see Table 1)by eliminating the inefficient immunoprecipitation and labor intensivelibrary preparation steps of the method and bringing high throughputsample processing and multi-analyte capabilities to the ChIP method.

TABLE 1 ChIP-Seq TAM CHIP-Seq Reagents Reagents Steps needed Time Stepsneeded Time Cell fixation 4 2 30 4 2 30 Chromatin 5 2 30 1 1 5preparation Shearing 6 0 20 0 0 0 ChIP reaction 40 20 48 hours 6 6  6hours Libraries 12 19  8 hours 3 3 1 hour 67 43 3-4 14 12 1 stepsreagents work days steps reagents work day

TAM-ChIP enables rapid (<24 hour elapsed time) and streamlined analysisof one or several protein-chromatin interactions for analysis of asingle gene all the way through to genome-wide interrogation. To achievethis, proteins, such antibodies specific for the protein(s) of interest,transcription factors, or chromodomains, such as in HP1 and Polycombproteins are first conjugated to a transposase:transposon complex(Transposome™) charged with synthetic oligonucleotide(s) that comprise atransposon cassette containing the following features:

Transposase recognition sequences required by the for catalysis of theDNA integration reaction;

-   -   a biotin (or other) molecule conjugated to an oligonucleotide,        preferably at one end, to facilitate purification of targeted        DNA with streptavidin magnetic beads (or other suitable support        conjugated to the other member of the selected high affinity        binding pair);    -   unique bar code sequences (i.e., short nucleotide sequences,        i.e., from 1-1,000 bases, preferably 1-50 bases, preferably        fewer than 20, even more preferably fewer than 10 bases) that        uniquely label an oligonucleotide species so that it can be        distinguished from other oligonucleotide species in the        reaction, and which correspond to a particular antibody) for        antibody identification in multi-analyte applications in which        multiple antibodies are used simultaneously with the same sample        material;    -   for whole genome sequencing applications, platform-specific tags        required for next generation sequencing (NGS).

In some aspects, rather than using a protein conjugated to aTransposome, the synthetic oligonucleotide described above will alsocontain sequences that are able to bind to non-coding RNA, suchsequences may include locked nucleic acids (LNA).

In still other aspects, rather than using a protein conjugated to aTransposome, the Transposome with synthetic oligonucleotide will beconjugated to a molecule that recognizes G-quadruplex structures, suchas small molecules and/or peptide nucleic acids (PNA). DNA-PNA chimericoligomers can be synthesized using techniques known in the art [42].

The antibody-transposase conjugates are incubated with chromatinfragments extracted from isolated cells, tissue, or whole organs (orother cell-containing biological samples) to allow specificantibody-protein binding. The transposase is subsequently activated byaddition of a cofactor, e.g., Mg²⁺, after sample dilution to preventinter-molecular events. Transposase activation results in insertion ofthe two transposase-associated oligonucleotides into the chromatin inproximity to the region where the antibody-associated DNA fragmentbound, thereby producing analysis-ready templates following adeproteination step and capture of biotin-tagged DNA fragments usingstreptavidin-coated magnetic beads.

Leveraging Tn5 Transposase for Improving ChIP

Transposable elements are discrete DNA segments that can repeatedlyinsert into a few or many sites in a host genome. Transposition occurswithout need for extensive DNA sequence homology or host gene functionsrequired in classical homologous recombination [15]. Consequently,transposable elements have proven to be superb tools for moleculargenetics and have been used extensively in vivo to link sequenceinformation to gene function. More recently, in vitro applications havealso been developed, specifically for Tn5, a class II “cut and paste”transposable element isolated from gram negative bacteria [16].Catalysis involves nicking of DNA to generate nucleophilic 3′ OH groupson both strands at the ends of the 19 bp Tn5 transposase DNA recognitionsequence. The 5′ ends are also cleaved within the synaptic complex,releasing the transposable element from the donor DNA (FIG. 3, Panel A).This mechanism allows for the formation of a stable complex between theenzyme and transposon in the absence of Mg²⁺[17], and is the basis forthe in vitro transposase technologies developed by EpicentreBiotechnology (Madison, Wis., USA).

Transposases are not conventional enzymes in the classical sense, inthat there is no turn-over. Spontaneous product release is not requiredand consequently the transposase is required in stoichiometricquantities [15].

Tn5-mediated transposition is random, causing a small 9 bp duplicationof the target sequence immediately adjacent to the insertion site (FIG.3, Panel B). The result is analogous to using a restriction endonucleasewith random sequence specificity that also contains a ligase activity.Epicenter's EZ-Tn5 Transposome™ technology utilizes atransposase-transposon complex which exhibits 1,000 fold greateractivity than wild type Tn5, achieved by combining a mutated recombinantTn5 transposase enzyme with two synthetic oligonucleotides containingoptimized 19 bp transposase recognition sequence [16, 18], and is thebasis of Epicentre's Nextera™ product used to streamline NGS librarypreparation. Using such a recombinant enzyme (whether naturallyoccurring or engineered to have improved transposition activity),transposition occurs with at efficiencies of 0.5-5%, using as little as50 ng of purified DNA, yielding >106 transpositions per reaction. Thetransposome is so stable that it can be introduced via electroporationinto living organisms, both prokaryotic (Gram negative and Gram positivebacteria [19-22]) and eukaryotic (yeast, trypanosome, and mice [19, 23,24]) where in the presence of endogenous Mg²⁺, transposon insertion hasshown to be random and stable. The ability of the Tn5 transposase torecognize eukaryotic chromatin as a substrate is extremely significant,as it can be adapted to transform ChIP into a multi-analyte methodsuitable for high through-put applications.

As described above and depicted in FIG. 1, Panel B, TAM-ChIP technologydevelopment uses an antibody-transposome linking moiety to effectivelyconjugate the Transposome™ to a targeting antibody that binds a targetedDNA-associated protein. Binding of the antibody to its target protein inchromatin (or other nucleic acids with which the protein associates incells under physiological conditions) optimizes transposase activitywith chromatin as a DNA substrate. The TAM-ChIP method involves allowingformation of complexes between the antibody-Transposome™ conjugate andchromatin fragments containing the antibody's target protein. Thesamples are then diluted (to ensure transposition of the oligonucleotidepayload in the transposon cassette into the same DNA fragment) and thechromatin-associated-transposase activated by the addition of the Mg²⁺co-factor, resulting in the insertion of the transposon cassettecontaining bar-code sequences and NGS compatible primer sites intoflanking DNA regions (FIG. 1, Panel B). Following DNA purification toremove proteins, PCR amplification (or another suitable amplificationprocess) with primers complementary to the oligonucleotides in the canbe performed to generate NGS compatible libraries for sequencing.

As described herein, the transposon is loaded with oligonucleotidescontaining both the transpose recognition sequences and sequences forsequencing on the Illumina platform. This enzyme-DNA complex (FIG. 9(a)) is called a transposome, and the resulting fragmented DNA intowhich the transposase has inserted its oligonucleotides is calledtagemented DNA.

Applicants investigated two forms of the Tn5 enzyme for testing inTAM-ChIP. One form of the enzyme was the same as that sold in theNextera kit (Tn5) while the second (TS-Tn5) was a temperature stablevariant under development by Illumina. Applicants initially determinedthe compatibility of chromatin extraction buffers with the transposaseenzymes and established that chromatin was recognized as a Tn5transposase substrate. FIG. 10 shows that assembledenzyme:oligonucleotide complexes (Panel A, Tn5, Panel B, TS-Tn5)completely tagment purified genomic DNA from HeLa cells (human cervicalcarcinoma), a cell line routinely used in ChIP experiments. Buffercompatibility experiments established that relative to the reactionbuffer provide in the Nextera kit, both transposase enzymes retainedfull activity in chromatin extraction buffers.

Also disclosed herein is the surprising discovery that sheering ofchromatin by sonication to achieve smaller, soluble fragments is notnecessary in TAM-ChIP. This unexpected result will further reducetechnical barriers and equipment needs required for ChIP and otherrelated techniques using the assisted transposon technology describedherein.

The direct insertion of the oligonucleotide duplex in the transposoncassette by the transposase eliminates the need for immunoprecipitation,thereby reducing the input DNA requirement. It can also eliminate theneed for ultra-high affinity antibodies, thereby expanding theapplication of the ChIP technique to a broader range of cellular targetswhich were previously excluded due to the lack of suitable antibodies.The inclusion of barcode sequences in the oligonucleotides allows forthe identification of the corresponding immunoprecipitating antibody,and is the basis of the multi-analyte potential of TAM-ChIP, which forthe first time enables simultaneous use of multiple antibodies in thesame sample and experiment. This innovation also has the benefits offurther reducing sample size requirements and enables elucidation ofprotein co-association in sequence-specific contexts throughout thegenome.

The construction of a functional antibody-transposome complex can bebased on a primary conjugation scheme or secondary conjugation scheme asdepicted in FIG. 11. Secondary antibody conjugates bind to primaryantibodies and can be used in conjunction with any primary antibody,while primary antibody conjugates bind directly to their targetedprotein. The DNA tag component of the primary conjugates can be changed,thus giving each primary conjugate a unique signature. Both approachesbring the TN5 Transposase enzyme into close proximity to DNA thusallowing DNA tagging to occur. Disclosed herein is a secondaryconjugation scheme. This was achieved by first conjugating a mixture ofthe two oligonucleotides (six-molar excess) to an anti-rabbit secondaryantibody such that a cleavable link between antibody and oligonucleotidewas generated. Size exclusion chromatography was used to separate theantibody-oligonucleotide conjugate (FIG. 12, lane 1) from unincorporatedoligonucleotides (FIG. 12, lane 2).

The functionality of this conjugate was tested in ChIP to confirm thatthe addition of oligonucleotides did not interfere with its ability tointeract with rabbit primary antibodies. A two to three fold reductionwas observed with the conjugate but was deemed not significant.

The ability of the antibody-tethered oligonucleotides to form afunctional transposome was also determined. Activity was first tested at55° C. for 30 min, the standard temperature for the Tn5 transposase, andalso for longer durations at 37° C. (FIG. 13) due to concerns thatelevated temperatures when applied to the TAM-ChIP procedure woulddestabilize antibody-chromatin interactions. Tagmentation of purifiedgenomic DNA was detected at both temperatures. However, a portion ofuntagmented input DNA (arrow) remained in all conditions indicating thatthe tagmentation was incomplete. It was noted that less untagmented DNAremained in all TS-Tn5 reactions, suggesting that theTS-Tn5-transposome-antibody complex may be more robust than its Tn5counterpart. However, it is not clear if the incomplete tagmentationreactions with the transposome-conjugates are due to reduced transposaseactivity, incomplete or incorrect assembly of the transposome complex ordue to steric hindrance from being antibody-tethered. However, theresidual activity is still sufficient for TAM-ChIP. Primary antibodytargeting of the transposome-antibody conjugate to chromatin couldovercome the decrease in random transposition activity through primaryantibody mediated stabilization of the transposome/antibody/chromatincomplex, which would effectively drive the reaction forward.

Data disclosed herein shows that the antibody-transposome constructcould be directed to chromatin in a specific manner through a primaryantibody that binds trimethyl-lysine at residue 4 of Histone H3(H3K4me3), a post translation modification found in the promoter regionsof transcriptionally active genes. FIG. 14 shows that, in the presenceof Mug of chromatin, but not 0.1 μg, the TS-Tn5 transposome complex butnot the Tn5 transposome complex effectively tagmented chromatin regionsbound by the primary antibody. Further, the untranslated regions onchromosome 12 and 20, showed no enrichment, indicating no tagmentationby the transposase, confirming that the primary antibody was directingthe transposase to chromatin fragments associated with antibody. Controlreactions, lacking either the primary antibody or lacking the secondarytransposome conjugate, showed no tagmentation in any of the regionsanalyzed. The no primary antibody control reaction confirms thattagmentation is driven solely by primary antibody binding events. Thesecondary antibody alone is unable to direct tagmentation of chromatin.

Next generation sequencing data from normal ChIP and from TAM-ChIP wasfound to be nearly identical as shown in FIG. 15.

As stated above, transposon targeting can be achieved utilizing not onlyantibody-transposon targeting, but also targeting via protein-proteinand/or protein-DNA interactions in which the transposon is conjugated toa protein that would target a protein-binding partner or protein-bindingdomain on the chromosome. Examples of such proteins include, withoutlimitation, methyl-binding proteins, proteins containing the followingdomains: bZIP domain, DNA-binding domain, helix-loop-helix,helix-turn-helix, MG-box, leucine zipper, lexitropsin, nucleic acidsimulations, zinc finger, histone methylases, recruitment proteins,Swi6. For example, conjugating a transposon onto MBD2, a protein thatbinds to the methyl group in DNA, would enable tagging chromatin DNAwith the specific DNA code of the transposon where MBD2 binds-allmethylated CpG binding sites (see Example 14). Similarly, bindingdomains, such as chromodomains [41], can be cloned into vectors andexpressed in the appropriate cells to create GST-fusions proteins, whichafter purification can be conjugated to the Transposome using themethods described herein or known in the art. These complexes could thenbe isolated using a GST-binding resin. For example, using chromodomainsfrom MPP8, CBX2, CBX7, ADD from ATRX and PWWP domain from DNMT3a wouldenable binding to H3K9me3, H3k27me3, H3K27me3, H3K9me3, and H3K36me3,respectively.

Transposon targeting can also be based on RNA-protein interactions,RNA-DNA interactions. For example, the TAM-CHIP methods described hereincan be modified to work with Chirp and CHART. Given the similarities toChIP it is possible to modify the TAM-ChIP protocol to work in Chirp andCHART procedures. The advantage of this approach would be in thesimplification of the Chirp and CHART protocol since library generationoccurs “automatically” using the transposase-targeted approach. A basicoutline of how this would be achieved is listed below.

-   -   1. A series of modified (biotin, streptavidin or other        attachment chemistry) oligonucleotides are designed to hybridize        to the ncRNA of interest.    -   2. Formaldehyde fixed chromatin is prepared (similar to what is        done for ChIP).    -   3. Oligonucleotides are hybridized to the native target within        the chromatin prep.    -   4. Transposase enzymes preloaded with a sequencing library        compatible transposon (similar to TAM-ChIP approach) and        modified with a complimentary attachment chemistry are added to        the material generated from Step 3 (above). These enzymes will        bind to the complimentary chemistry on the hybridized oligos        resulting in the intended targeting of the transposon to the        sites of interest.    -   5. Following washing, magnesium is added to activate the        transposase resulting in the integration of library adaptors to        the sites of interest.    -   6. PCR amplification results in sequence-ready libraries    -   7. DNA is sequenced using Next-Gen sequencing platforms such as        Illumina resulting in the genome-wide identification ncRNA        interaction sites.    -   8. As an alternative to steps 3 and 4, the biotinylated or        otherwise chemically modified oligos could be preincubated with        the streptavidin or otherwise chemically modified transposase        complex. This entire complex could then be hybridized to the        chromatin sample.

Methods and Representative Examples

Preferred methods, materials, and conditions for carrying out somepreferred, non-limiting, representative embodiments of the invention aredescribed below. Those of ordinary skill in the art will readilyappreciate that the invention can be practiced in a number of additionalembodiments using equivalent alternate techniques and materials.

Example 1 TAM-ChIP Preliminary Data

In order to improve the turnaround-time of conventional ChIP-Seqservices, Epicentre's Nextera™ DNA Sample Prep kit, which uses theEZ-Tn5 Transposome™ and suppression PCR to generate NGS compatiblelibraries, was evaluated for suitability for use with ChIP-enriched DNA.ChIP was performed in duplicate using p53 antibodies and 30 μg chromatinextracted from estrogen stimulated MCF-7 cells (a human breast cancercell line) following established protocols, and isolated DNA was thenpurified. Quantitative PCR was performed on known p53 binding sites tovalidate the specificity of the anti-p53 ChIP reactions (FIG. 4, PanelA). Untr12 is a negative control in a gene desert on human chromosome 12and is not expected to be bound by p53.

The Nextera transposition reaction was performed using two quantities ofChIP DNA (FIG. 4, Panel B) according to the manufacturer's protocol. TheDNA libraries were purified and used for PCR according to the Nexteraprotocol for 18 cycles. The amplified DNA was purified and quantified bymeasuring absorbance at 260 nm (A260) using a NanoDropspectrophotometer. The amount of DNA produced in the Nextera reactionwas in the range of what is typically obtained using the Illuminalibrary protocol.

These data demonstrate the suitability of EZ-Tn5 for use with fragmentedDNA substrates, and that the p53 binding sites detected in traditionalChIP are preserved and quantifiable in Nextera-generated libraries.Interestingly, a higher amount of DNA was generated in the Nexterareaction with the smaller amount of DNA isolated by ChIP, suggestingthat the transposition efficiency was higher and that less inputchromatin may be required for ChIP experiments when EZ-Tn5 isincorporated into the methodology.

For the methods described below, the EZ-Tn5 transposome is purchasedfrom Epicentre Biotechnology (Madison, Wis., USA) and ChIP-IT Express™reagents and protocols are used (Active Motif, Carlsbad, Calif., USA) asthe ChIP reagents throughout this example. The end result is anoptimized method for the ChIP-validated antibody-transposome conjugates.

The methods below are performed in human Hela cell lines, which areeasily cultured in vitro to produce the necessary quantities of genomicDNA (gDNA) or chromatin required for the experiments described below.While many epigenetic research tools and consumables target researchersusing vertebrate animal model systems, largely because this segment isthe largest in the epigenetic research tools market, the principleepigenetic mechanisms are conserved throughout vertebrates (includingthe primary amino acid sequence of histones and the repertoire ofpost-translational modifications), although those skilled in the artwill be able to adapt the reagents and methods of this invention for usewith other organisms. Another compelling reason for the use of mammaliancells for the TAM-ChIP technology stems from the complexity of thegenome. ChIP is far more challenging in mammalian cells, where genesrepresent only 1-1.5% of the genome, than in lower eukaryotes wheregenes represent a much large fraction of the total genome (compare with70% in S. Cerevisiae).

TABLE 2 Candidate HeLA genomic loci for qPCR analysis of transpositionefficiency Transcriptionally Active GAPDH HOX10 EEF1A1 TUB1C LDHA RASSF1A ACTB PPIB PABPC1 RPS18 Transcriptionally Repressed PTGER3 HOXD13 HBBUntr12 NGB CFDP1 Sat2A MyoD PAX2 MYT1

Analytic Methods

The majority of the experiments described below require determination oftransposition efficiency, and evaluation of the distribution (bothabundance and range) of DNA fragments generated as a consequence oftransposition. Transposition efficiency can be determined using anysuitable technique, for example, by quantitative real-time PCR using aStepOnePlus RT-PCR thermocycler (Applied Biosystems) and primerscomplimentary to a panel of genomic loci known to be eithertranscriptionally active or repressed in Hela cells (Table 2, above)[25]. Transposition results in the insertion the biotin-taggedtransposon oligonucleotide into the target DNA, enabling isolation oftransposon-tagged DNA fragments with streptavidin-coated magnetic beadsand subsequent quantitation in triplicate by real time PCR. A five-folddilution series of fragmented Hela genomic DNA can be used as standardsto generate a quantitation curve. Identical locus-specific PCR primersets are used for both samples and standards, and transpositionefficiency will be calculated as the median of the DNA recovered for allloci. The generation of tagged fragments less than about 200 bp isparticularly preferred to achieve the necessary resolution of sequencereads in NGS applications. Evaluation of the abundance and range oftransposon tagged-DNA fragment sizes produced by transposition eventsrequires, for example, an Agilent 2100 Bioanalyzer, which employs amicrofluidics system for electrophoretic determination of size andquantity of DNA fragments in sample volumes of 1-4 μl.

Example 2 Antibody-Transposase Conjugates

TAM-ChIP requires that the enzymatic activity of the transposasepreferably be unaltered, with regards to catalytic rate and randomnessof integration sites, when coupled to another protein. Conjugations withvarious chemistries and crosslinkers of varying length are comparedusing ChIP validated antibodies. This example generates functionalantibody-transposome conjugates.

An extensive number of ChIP-validated antibodies are commerciallyavailable or can be developed using conventional antibody productiontechniques. Here, antibodies to a chromatin associated protein (RNApolymerase II) and a structural chromatin protein, a histone(anti-histone H3 trimethyl-lysine 4 (H3K4tm) mark associated withtranscriptionally active chromatin), are conjugated to the EZ-Tn5transposome using any suitable approach, two of which are describedbelow.

Antibodies can be chemically crosslinked either to the transposase(protein-protein) or to the transposon (protein-DNA) using HydralinkChemistry (Solulink, San Diego, Calif., USA), which isstoichiometrically more efficient than traditional EDC/NHS chemistriesand has been used in the development of PCR-based proximity ligationassays, recognized as the most sensitive assay for proteindetection[26-28]. The chemistry involves formation of reaction betweenan aromatic hydrazine (hydrazinonicotinamide-HyNic) and an aromaticaldehyde (4-formylbenzamide-4FB), yielding a stable bis-arylhydrazonethat is UV-traceable, absorbing at 350 nm Conjugation reaction kineticscan be augmented 10-100 fold in the presence of aniline, leading toconjugation yields of >95%[26].

Conjugations are performed following the manufacturer's establishedprotocols in quantities sufficient for their functional characterizationdescribed below and for their subsequent use in the methods described.Both antibody-transposase and antibody-transposon, thetransposase-associated oligonucleotide (FIG. 6) conjugates is preparedusing varying crosslinker lengths (0, 6 or 12 carbon side-chains) forprotein-protein conjugates or transposon oligonucleotides of varyinglengths (synthesized with additional 20, 40 or 60 bp), with the 4FBmoiety incorporated during solid phase synthesis. Conjugatestoichiometry is determined by measuring absorption of thebis-arylhydrazone crosslinking product at 350 nm which has a molarextinction coefficient of 1600 M·l [29]. Aliquots reserved at each stepare used to monitor transposase activity by measuring transpositionefficiency with lambda DNA (as above) and retained antibody recognitionof antigen by dot blot analysis using established Active Motifprotocols. This Example provides isolation of antibody-transposaseconjugates with a stoichiometry of greater than or equal to twotransposase molecules per antibody molecule in which the function ofantibody and transposase is no less than 90% of their unconjugatedcounterparts. These conjugates are used below for the TAM-ChIPtechnology. Methods for conjugation of antibodies to a variety ofmolecules (enzymes, dyes, oligonucleotides, biotin) are well establishedand are considered routine. Tn5 transposase fusion proteins have beendescribed and are functional [30, 31]. Accordingly, any suitableapproach can be adapted for use in the context of this invention.

Example 3 TAM-ChIP Optimization

Examples 1 and 2 above provides the basis for performing TAM-ChIP anddemonstrating its benefits relative to traditional ChIP methods. Theoptimized chromatin extraction and fragmentation procedure above iscombined with the antibody-transposome conjugate to perform the TAM-ChIPprocedure. A method of comparing the genomic representation of thesequencing libraries produced by TAM-ChIP and traditional ChIP-Seq isalso provided. This is done using two steps. The first step involvesoptimizing sets of conditions with regards to chromatin andantibody-transposase concentrations, optimization of incubation timesusing transposition the analytic methods describe above as the readout.The second step is a direct comparison of the genomic representation ofthe DNA libraries produced by TAM-ChIP with that of conventionalChIP-Seq methods.

An optimal protocol can be determined using the steps depicted in FIG.7. First, the optimal amount of chromatin substrate is determined, asthis impacts both transposition efficiency and fragment size. Initially,antibody-transposase conjugates are used at a fixed amount, where in theamount of transposase enzyme present corresponds to the amountrecommended in applications developed by Epicentre Biotechnology for usewith 50 ng DNA.

Triplicate samples of 50, 150, and 450 ng of Hela cell chromatin(quantitated by A260) are incubated with the antibody-transposaseconjugate in 100 IJI for two hours at 4° c. (FIG. 7, column 1). Thechromatin-antibody complexes are diluted in LMW buffer and transposaseactivated by the addition of 10 mM Magnesium acetate using the optimizedtransposase reaction conditions developed above. Samples are treatedwith 201 μg Proteinase K for 1 hour at 37° C. and DNA purified using aZymo DNA Clean & Concentrator-5 Kit (or equivalent). If formaldehydecrosslinked chromatin is used, prior to protease treatment, reversal ofcrosslinks is achieved by the addition of an equal volume of ReverseCross-Linking Buffer (50 mM NaCl in 50 mM Glycine) and samples incubatedat 95° C. for 15 min

Biotin-tagged DNA fragments are captured using streptavidin magneticbeads and transposition efficiency and fragment size profiles aredetermined as described above. Transposition efficiency is significantlyhigher at the transcriptionally active genomic targets listed in Table 1than at the transcriptionally silent regions that are analyzed by qPCR.Consequently, for these experiments transposition efficiency iscalculated as a relative ratio of transposition into transcriptionallyactive and inactive regions, thereby providing a means for comparison ofthe specificity and efficacy of the antibody-transposome complexes. Therange of input chromatin is expanded in subsequent experiments iftransposition efficiencies are too low or tagged-DNA fragments toosmall, the latter a consequence of too little DNA. This set ofexperiments identifies the antibody-transposome conjugates with optimalactivity for chromatin substrates and which chemistry is optimal for thegeneration of additional antibody-transposase conjugates, such as anon-immune IgG-transposase negative control required for the TAM-ChIPprotocol described below.

The optimal conjugate for each of the two antibodies (RNA polyermase IIand H3K4tm) is used in the following subsequent experiments (FIG. 7,columns 2 through 4) designed to optimize the effects of differentantibody-transposase concentrations, antibody-chromatin incubationtimes, and sample dilution on transposition efficiency and fragmentsize. The conditions yielding optimal results are carried forward in thesubsequent rounds of procedure optimization. Antibody-transposaseconcentrations are varied in a two-fold dilution series consisting of2×, 1× and 0.5×; incubations of chromatin with antibody-transposaseconjugates are varied for 0.5, 1, 2, and 4 hours; and sample dilutionprior to transposase activation to ensure intra-complex transpositionare varied as five-fold dilution series (1:×; 1:5×; 1:25×, and 1:125×,where × represents the minimal dilution factor determined using themethods described herein). The ranges of variables are expanded aswarranted based on observed fragment size and transposition efficiency.These experiments identify the conditions which produce a minimum of 500ng of <200 bp tagged-DNA fragments following 18 cycles of PCR-amountsrequired for the Illumina sequencing platform. These experiments resultin an optimized TAM-ChIP methodology.

Example 4 Validation of NGS Libraries Generated by TAM-ChIP

The DNA libraries produced by the optimized method in developed in thepreceding experiments with IgG, RNA polymerase II, and H3K4tmantibody-transposome conjugates are compared with the libraries producedvia traditional ChIP-Seq performed with the same unconjugatedantibodies. For traditional ChIP-Seq, Hela chromatin extracts generatedfor the above set of experiments are incubated with 5 μg antibody for 16hours at 4° C. 1 μg are left unprocessed and serve as the input control.Antibody-chromatin complexes are captured using protein A coatedmagnetic beads, washed, eluted, and DNA purified following establishedprocedures. ChIP with 5 μg of non-immune rabbit IgG is performed inparallel as an antibody specificity control. The ChIP-enriched and theuntreated sonicated gDNA are processed according standard protocols forlibrary preparation for sequencing in the Illumina Genome Analyzer GAll.This consists of end-repair, adaptor ligation, size-selection and PCRamplification, and all these steps are done and sequencing performedaccording to standard methods. The generated data from both TAM-ChIP andtraditional ChIP from two independent experiments is analyzed. Readsmapped to the human genome (alignments) are analyzed to find genomicregions with significant enrichments (“peaks”) over alignments obtainedfrom either

Input or IgG control DNA. Dozens of H3K4tm and RNA Polymerase IIChIP-Seq assays are performed and analyzed, and very similar results areobtained with the peak calling algorithms MACS [32], SICER [33], or CCAT[34]. In addition, software is used to extend the read alignments to theactual length of the DNA fragments (−200-250 bp), and to generate a“signal map” showing alignment (“tag”) densities in 32-bp bins acrossthe genome and reproducibility between replicates is typically −80%.Peaks and signal maps are entered into gene annotation and samplecomparison software, returning concise Excel tables showing peak metricsand location of peaks relative to genes. These are used to compare therepresentation of genomic sequences in the DNA libraries prepared by twomethods and show concordance of genomic coverage.

Example 5 Additional TAM ChIP Embodiments

The methods established above will be recognized by those of ordinaryskill in the art to be readily carried out in other embodiments, e.g.,(a) those comprising antibodies from different animal hosts (rabbit,mouse, rat and goat) specific for proteins associated with eithertranscriptionally active euchromatin or transcriptionally silencedheterochromatin (i.e. HP1 proteins, and heterochromatin-associatedhistone marks), (b) TAM-ChIPs wherein antibody-transposase conjugatesare be used singly or simultaneously, and with different degrees ofcomplexity (two-plex, three-plex, etc.), including versions with eachconjugate bearing a unique bar-code sequence for antibodyidentification, (c) those where the antibody-oligonucleotide conjugatesprepared above are used in a multiple proximity ligation method (see,e.g., Example 6, below). Antibody-oligo conjugates bound to chromatinare diluted, followed by proximity ligation of the antibody-associatedoligonucleotide with the associated chromatin fragment end and nickssealed. Ligation of oligonucleotides to chromatin has been used to mapchromatin higher order structures [35], where co-associating chromatinends in isolated complexes containing higher-order structures are taggedvia ligation with primers and then ligated to each other via theirproximity, supporting the feasibility of this approach. Use of areversible antibody-oligonucleotide crosslinking chemistry or theinclusion of a rare restriction endonuclease cleavage site allowsliberation of the antibody from the DNA now tagged with the bar-codecontaining oligonucleotide which is then directly amplified for NGSusing an appropriate PCR amplification strategy.

Example 6 Antibody-Oligonucleotide Conjugates and Proximity Ligation

These methods use cross-linked and sonicated (or restriction digested)chromatin as a starting material. Instead of conjugation to transposase,this approach uses conjugation of an antibody to short double-strandedDNA oligonucleotides of known sequence. The conjugate is incubated withcross-linked chromatin that has been either restriction enzyme digestedor sonicated, resulting in antibody binding at the intended target.Proximity-mediated ligation is performed, resulting in ligation of theantibody delivered oligos to the target-associated free genomic DNA ends(FIG. 8). Digestion of the cross-linked chromatin and the proximityligation strategy using sonicated or restriction enzyme digestedcross-linked chromatin is well established, and these methods build uponand inventively improve on those used in the 5C and Hi-C technologies(Dostie and Dekker, 2007; Lieberman-Aiden et al., 2009). The key to theligation is to perform this step under diluted conditions which favorthe interaction of DNA molecules held in close proximity. Followingligation, crosslinks can be reversed by heating to 65° C. overnight, andproteins can be removed with proteinase K treatment. The regions ofinterest (e.g., the regions bound by the protein of interest andtargeted by the antibody) can be enriched by PCR using primers thatanneal to regions within the ligated oligonucleotide sequence, and theresulting amplified DNA can be sequenced using, for example, theIllumina platform, resulting in a genome wide protein binding profile.In addition, this approach is well suited to generate binding profilesof multiple factors in the same sample. This is achieved by designingmultiple oligonucleotides, each containing a unique bar code sequence,and conjugating these unique oligos to different antibodies. Multipleantibody conjugates can be added to the same sample at the same time.After sequencing, the data for the multiple targets can be sorted basedon the bar code sequence.

Oligonucleotide Embodiments.

Several features can be designed into the oligonucleotide(s) that areconjugated to the antibody(ies). These features are listed below anddepicted in FIG. 8.

1. The oligonucleotide is double-stranded and the 5′ end of one of thestrands is linked to biotin (or a member of different high affinitybinding pair). The biotin is used for conjugation to the antibody.2. There is a restriction site (e.g., Not 1, a “freeing” restrictionenzyme in the context of the invention) encoded in each oligonucleotideto allow the oligonucleotide to be separated from the antibody, ifneeded.3. There is a region of sequence included that functions only for thepurpose of varying the oligonucleotide length. The ligation of theoligonucleotide to the free genomic ends of the captured DNA may bedependent on the length of the oligonucleotides. The entireoligonucleotide is typically about 80 nucleotides in length, althoughlonger or shorter lengths may be optimal in a given application.4. A region is included that is complementary to Illumina (or othersuitable) primers. This region facilitates amplification ofoligonucleotide-ligated genomic DNA, preferably to be compatible withsequencing on the intended (e.g., Illumina) platform.5. There is a 4-base pair (or shorter or longer) barcode. Severaldifferent oligonucleotides can be synthesized, each having a differentbar code. Oligos with different bar codes can be conjugated to differentantibodies, thus allowing multiple antibodies to be used in the samereaction.6. There is a restriction-site-compatible overhang that allows theoligonucleotide to be ligated to restriction-digested genomic DNA. Theoverhang may preferably be a 4 nucleotide overhang (e.g., GATC, which iscompatible with Dpn II, Mbo I, and Sau3A I, digestions). In such cases,the genomic DNA is cut with a restriction enzyme that having a 4 bprecognition site, which should on average cleave the DNA every 256bases. Alternatively, a combination of restriction enzymes having 6 bprecognition sites can be used. Alternatively, TA cloning can be used. Insuch embodiments, sonicated DNA is used which has gone through endrepair and A overhang addition. The oligonucleotides are designed tohave T overhangs.

Example 7 Alternate Antibody/Oligonucleotide Conjugation Embodiments

Any suitable chemistry can be used to achieve theantibody/oligonucleotide conjugations used in this invention. One suchapproach is described below.

1) The biotinylated forward strand oligonucleotide is annealed to theunbiotinylated reverse strand using standard procedures.2) The antibody can be biotinylated using a number of available kits,for example, the Solulink Chromalink One-Shot biotinylation kit, whichallows for quantitation of the number of biotins per antibody and thusallows for optimization of the number of biotins conjugated to theantibody.3) Self-assembly of the conjugate can be achieved by mixing appropriateratios of the biotinylated oligo, biotinylated antibody, and freestreptavidin, a tetra mer with four biotin binding sites all of whichcan be simultaneously occupied.4) Unconjugated antibody and oligo can be removed using streptavidinmagnetic beads.

This approach has been used and validated using a ratio of 2:1:2 (oligo:free streptavidin:antibody). An anti-Goat IgG antibody was coupled to a100 bp oligo by mixing in the presence of free streptavidin. A goatantibody serves as the antigen and was absorbed to maxisorp 96-wellplates at different concentrations. The antibody/oligionucleotideconjugate was allowed to bind the antigen and excess antibody was washedaway. After washing, signal was detected using PCR with primers thatanneal within the conjugated oligonucleotide (FIG. 36).

Additional Examples Example 8 Compatibility of Chromatin ExtractionBuffers with the Transposase Enzyme and Determination of WhetherChromatin is Recognized as a Tn5 Transposase Substrate

For this analysis two forms of the Tn5 enzyme were used. One form of theenzyme was the same as that sold in the Nextera kit (Tn5) while thesecond (TS-Tn5) was a temperature stable variant under development byIllumina. FIG. 16 shows that assembled enzyme: oligonucleotide complexes(Panel A, Tn5, Panel B, TS-Tn5) completely tagment purified genomic DNAfrom HeLa cells (human cervical carcinoma), a cell line routinely usedin ChIP experiments. Buffer compatibility experiments established thatrelative to the reaction buffer provided in the Nextera kit, bothtransposase enzymes retained full activity in chromatin extractionbuffers.

Example 9 Identify the Minimum Dilution Factor with which theAntibody-Chromatin Complex Must be Diluted Such that TransposaseEfficiency is Unaffected

The lysis buffer used to extract chromatin from cells for the ChIPmethod contains harsh detergents such as SDS (for efficient extraction)and EDTA (to inhibit nuclease activity). In TAM-ChIP,antibody-transposase complexes are directly added to chromatin in lysisbuffer. Initial experiments were aimed at determining the minimumdilution factor of the chromatin in lysis buffer required to preservefull transposase activity is retained. A mock-ChIP experiment usingActive Motif's ChIP lysis buffer was performed to reproduce the buffercomposition present at the chromatin immunocapture step. This buffer wassequentially diluted two fold (ranging from undiluted to a 1:20dilution) into sterile water. Transposase was added to each bufferdilution with either 50 ng of unmethylated lambda phage DNA (48.5 kb,Promega) or 1 μg of genomic HeLa DNA. FIG. 17 shows the heterogeneouspopulation of DNA fragments produced by the random activity of thetransposase which appears as a smear following agarose gelelectrophoresis. The enzyme “tagmented” (defined as fragmentation ofsubstrate DNA fragment through the cutting and insertion of appended-endoligonucleotides by the transposase) both DNA templates equally well,even in undiluted chromatin extraction buffer conditions. These dataindicate that the chromatin extraction buffer does not inhibit thetransposase and that no dilution of the chromatin will be needed at thisstep of the TAM-ChIP procedure. Thus, alteration or optimization ofchromatin extraction buffer formulation is not required. We observedthat the range of fragment sizes produced by the transposase differedfor the two DNA templates. Lambda DNA generated higher molecular weightfragments relative to HeLa DNA. Consequently, use of the Lambda DNA as areference DNA or positive control was discontinued for subsequentexperiments. It was felt that HeLa genomic DNA would serve as a morerelevant control for subsequent experiments where chromatin is used.

Example 10 Confirm Activity of Custom-Order Tn5 and TS-Tn5 TransposomeComplexes

Applicant manufactures all buffers, and reagents as part of itsdevelopment process. To confirm that the efficiency of the transposasetagmentation reaction was equivalent with the use of Applicant'scomponents compared to those provided in the Illumina Nextera kits.A-METS and B-METS oligonucleotides corresponding to the Illuminasequences were ordered (IDT, Inc.) and were annealed to p-MENTS (FIG.18) to form the double stranded DNA (with appended ends) as thetransposase will only bind its recognition sequence in the context ofdouble-stranded DNA. Equal ratios of single-stranded oligonucleotides A-or B-METS together with p-MENTS were incubated at 98° C. for two minutesfollowed by 60 cycles of decreasing temperature of 1.3° C. per cycle.The concentration of the annealed oligonucleotides was determined on aNanodrop spectrophotometer. The annealed oligonucleotides were analyzedon a 3% agarose gel to ensure complete annealing. Next, transposomeswere assembled using both Tn5 and TS-Tn5 enzymes and the annealedoligonucleotides. Assembly occurs spontaneously in a one-hour incubationat room temperature at a 1:1 molar ratio. To confirm that thesetransposome complexes generated in house would tagment the DNA with thesame efficiency as the EZ-Tn5 product provided in the Nextera DNA samplepreparation kit (used in FIG. 18), varying amounts of Tn5 and TS-Tn5transposomes were incubated with 1 μg of genomic HeLa DNA for 5 minutesat 55° C. and 250 ng of the tagmented DNA were analyzed on a 1.5%agarose gel. The efficiency of the newly assembled transposome complexeson genomic HeLa DNA was confirmed, with increased amount of enzymeresulting in increased tagmentation (FIG. 19), producing a largerpopulation of DNA fragments of smaller size.

FIG. 20 shows that Applicant's tagmentation buffer performed as well asthe Illumina buffer provided in the Nextera DNA sample preparation kit.

It was noted that unintegrated oligonucleotides were visible in theagarose gels in FIGS. 19A and 20, raising concern that either not allenzyme was loaded with olgios or that the free oligonucleotides couldinterfere with transposase activity. To address these concerns, Tn5 andTS-Tn5 transposases were assembled with double stranded oligonucleotideswith increasing amounts of enzyme, with ratios ranging from 1:1 to 1:100(oligonucleotide:transposase) and tagmentation reactions preformed withgenomic HeLa DNA were visualized on a 1.5% agarose gel (FIG. 21). Whileincreasing amounts of enzyme did decrease the amount of freeoligonucleotide, decreased tagmentation was detected at ratios of 1:10to 1:100. Ratios of 1:1 and 1:2 were the most effective in tagmentationas judged by the absence of high molecular weight input DNA in theseconditions, indicating that high concentrations of oligonucleotide arerequired to drive efficient transposome assembly. 1:1 ratios wereadopted as the standard condition for subsequent experiments.

The Illumina sequencing platform requires the addition of index primerscontaining bar-code sequences as well as other platform-specificfeatures to the tagmentation reaction products. These bar-codes allowfor of up to 96 distinct samples to be sequenced simultaneously. Theseprimers are added followed by a limited-cycle four-primer PCR reaction,the result of which are fragments that serve as input material forbridge PCR which generates the sequencing ready DNA library (FIG. 9).Since TAM-ChIP technology also relies on the barcoding strategy toachieve multiplexing capability, Applicants created new customizedadapter and primer sequences compatible with the Illuminaoligonucleotide sequences (A-METS and B-METS) and the Illuminasequencing platforms (Table 3).

TABLE 3 Adapter and Primer sequences for limited-cycle PCR Name SequenceAdapter 1a 5′-AATGATACGGCGACCACCGAGATCTTAAGGCG SEQ ID NO: 1ATCGTCGGCAGCGTC-3′ Adapter 2A 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTGTSEQ ID NO: 2 CTCGTGGGCTCGG-3′ Adapter 1b5′-AATGATACGGCGACCACCGAGATCTACACTCG SEQ ID NO: 3 TCGGCAGCGTC-3′Adapter 2b 5′-CAAGCAGAAGACGGCATACGATAGATCGCGTC SEQ ID NO: 4TCGTGGGCTCGG-3′ Primer 1 5′-AATGATACGGCGACCACCGA-3′ SEQ ID NO: 5Primer 2 5′-CAAGCAGAAGACGGCATACGA-3′ SEQ ID NO: 6

To test the compatibility of the in house designed oligonucleotidesassembled into transposomes with the Illumina Associated Nextera Indexkit, 100 ng of genomic DNA was tagmented by the in house assembled Tn5and TS-Tn5 transposome complexes, and subjected to limited-cycle PCRusing the four primers provided in the Illumina kit. FIG. 22 illustratesthe successful amplification of the tagmented genomic HeLa DNA with theNextera Index kit primers indicating compatibility between the variousprimers. Further, by increasing the amount of transposomes we were ableto obtain a population of smaller fragment sizes. Note however, that theDNA fragments are largely >200 bp in length which may reflect a spacinglimitation of the transposase enzyme.

Since the primers listed in Table 3 were based on sequences deduced formthe literature and not provided by Illumina, the next experiment wasperformed to validate this set of in house primers. Two differentversions of adapters 1 and 2 (called a and b) were synthesized (IDT,Inc). Different combinations of the primers and adapters provided byIllumina and the primers and adapters made by Applicant were used in PCRamplification of 100 ng tagmented genomic HeLa DNA. As depicted in FIG.23, the different combinations of the of the new adapters at the 5×concentration (2.5 μM of Adapters 1a and 2a, or 1b and 2b) together withthe new primers (50 μM of Primers 1 and 2) yield the same amplificationas the PCR sample containing only the adapters and primers provided inthe Illumina kit (FIG. 23, lane designated C+). Together these resultsconfirm that oligonucleotides listed in Table 3 are functional,compatible with the Illumina Associated Nextera Index kit and thereforesuitable for sequencing on the Illumina platform. In subsequentexperiments where the four primer PCR reaction is performed, the 1b and2b Adaptors are used at 2.5 μM with 50 μM of Primers 1 and 2.

Example 11 Confirm Activity of Tn5 and TS-Tn5 Transposome Complexes onChromatin

With Tn5 and TS-Tn5 activity on naked genomic DNA confirmed, the nextset of experiments were aimed at verifying the activity of the Tn5 andTS-Tn5 transposases on chromatin. Formaldehyde crosslinked HeLachromatin was mechanically sheared with Applicant's EpiShear ProbeSonicator with a cooled platform to generate chromatin fragments of lessthan 1 kb as per traditional ChIP protocols. 10 μg of chromatin wasincubated with different amounts of Tn5 and TS-Tn5 transposomecomplexes. Initial experiments were performed with transposomeconcentrations ranging between 5 and 20 units, amounts of enzymesufficient to tagment purified genomic DNA (data not shown). However, noor very low levels of tagmentation was detected with these amounts ofenzyme when the reaction products were analyzed by agarose gelelectrophoresis. These results suggested that the efficiency of thetransposase on crosslinked chromatin is significantly lower, so higherenzyme amounts were used in subsequent experiments. Up to 100 units wereutilized, and reaction products amplified by limited cycle PCR prior toagarose gel electrophoresis. In order to detect any tagmentation ofchromatin, the number of limited cycle PCR cycles was increased from 5to 25, and half of the PCR reaction resolved on the agarose gel (FIG.24A). The remaining half was purified using Agencourt AMPure XP magneticbeads to remove un-integrated adapters and primers and 500 ng resolvedby agarose gel electrophoresis (FIG. 24B). This material represents aDNA library suitable for use in NGS. Note in FIG. 24A that tagmentationof chromatin was successful in both unsheared and sheared conditions. Ithad been assumed that unsheared genomic DNA was too viscous foreffective in vitro manipulations. However these results indicate thatthe transposome complex is able to tagment even unsheared chromatin.Further, the majority of fragments in the population range between200-300 bp in length (Compare fragment sizes in FIG. 24 with those inFIG. 22), an ideal size for NGS library construction).

The above data establish that the transposase enzyme retains function inthe buffers that are used to extract chromatin from the nuclei ofmammalian cells and that both naked DNA and formaldehyde-crosslinkedchromatin do indeed serve as substrates, albeit the latter withsignificantly reduced efficiency. In addition, in house generatedtagmentation buffer, assembled transposomes and PCR primers fordownstream library amplification appear functionally as robust asreagents provided in the Illumina products.

Relative to pure DNA, tagmentation of chromatin does require moreenzyme; however, the end product—the range of DNA fragment sizegenerated—are essentially identical (compare FIGS. 22 and 24).Interestingly the size of the starting chromatin (whether fragmented bysonication or intact) did not impact enzyme efficiency, an unanticipatedresult that will translate into a shortened the TAM-ChIP procedure. Thisresult is suggestive that crude cell lysates containing chromatin,rather than isolated chromatin could be used as transposase substrates.

DNA fragments of less than 200 bp in length are typically used in thetraditional method of NGS library preparation where DNA fragments of100-200 bp in length are excised from an agarose gel. In all of the datashown above, whether chromatin or purified genome DNA was used asubstrate, the majority of the fragments produced were larger, in the200-300 bp range. This product range was also observed in positivecontrol samples where only Nextera kit reagents were used. This fragmentsize may represent threshold for Tn5 when presented with larger DNA (1kb or larger) substrates. The fragment sizes produced throughtagmentation are still very suitable for use in NGS sequencing. Whilethe larger fragment size could impact resolution of the sequencingreads, this could be compensated for by the randomness of the Tn5mediated insertion events, and could result in adequate genomiccoverage.

Example 12 Development of Antibody-Transposome Conjugates

Development of TAM-ChIP requires that the enzymatic activity of thetransposase is retained, with regards to catalytic rate and randomnessof integration sites, when coupled to an anti-rabbit secondary antibody(Jackson ImmunoResearch Laboratories Inc), as the conjugation partner(FIG. 11). Such a conjugate would be useful as a common reagent that canbe used for all rabbit antibodies with diverse specificities (histonemarks associated with open or closed chromatin, DNA damage, etc.)

Optimal Ratio of Primary to Secondary Antibodies.

In many applications secondary antibodies are used in excess of theprimary and multiple secondary antibody molecules can bind a singleprimary antibody molecule, thereby providing signal amplification.However, these conditions may or may not be ideal for TAM-ChIP. Initialexperiments were focused on establishing the optimal ratio of primary tosecondary antibodies using quantitative PCR of captured DNA as theanalytical method. Chromatin was prepared essentially as describedherein and Applicant's ChIP validated rabbit polyclonal specific fortrimethyllysine at residue 4 of histone H3 (H3K4me3, Catalog No. 39915)was used in ChIP with varying amounts of secondary IgG (JacksonImmunoResearch Laboratories Inc). Following enrichment, qPCR wasperformed using primers against untranslated region 12 (Untr12) andGAPDH. Untr12, a gene desert on chromosome 12, shows no or low H3K4me3enrichment, while GAPDH, an actively transcribed gene that is associatedwith the presence of H3K4me3, shows varying enrichment depending onamount of secondary antibody. The optimal ratio of primary H3K4me3antibody to secondary IgG is 1:1 with half the amount of primaryrelative to secondary (2:1) also giving a high signal (FIG. 25).

ChIP data is expressed as binding events detected per 1,000 cells whichrepresents the average of the raw data triplicates adjusted for theamount of chromatin in the reaction, the resuspension volume and theprimer efficiency. Applicant's custom ChIP service has performed andanalyzed hundreds of ChIP assays with a broad range of primaryantibodies and this calculation provides consistency in data analysisand allows direct comparison across samples and experiments. This scalecan be converted to enrichment over input (used in some figure below) bydividing binding event values by 1,000. Antibody-OligonucleotideConjugation.

The secondary antibody was conjugated to single-strandedoligonucleotides (A and B, Table 4) with appended ends containing at the5′ end two iterations of an 18-carbon long hexaethyleneglycol spacer(Sp18) and a 5′ Thiol Modifier C6 S-S (ThiolC6) viathiol-maleimide/thiol exchange chemistry which targets introducedmaleimide/thiol exchange residues on the antibody. The objective was tointroduce a cleavable (disulfide) bond between the antibody andoligonucleotide to facilitate separation and isolation of tagmented DNAfragments in downstream steps.

TABLE 4 Sequences of single stranded oligonucleotide A and B conjugated to a 18-carbon spacer Oligonucleotide AThiolC6//Sp18//Sp18/TCGTCGGCAG SEQ ID NO: 7 CGTCAGATGTGTATAAGAGACAGOligonucleotide B ThiolC6/Sp18//Sp18/GTCTCGTGGGC SEQ ID NO: 8TCGGAGATGTGTATAAGAGACAG

Conjugations were performed using a 6-fold molar excess ofsingle-stranded oligonucleotides to achieve antibodies containing aminimum of 2 oligos/antibody. In the initial approach, an equimolarmixture of A and B oligo was used in the conjugation reaction.Consequent, reaction products will contain a mixture of conjugationproducts (A:A, A:B and B:B). To create a cleavable link between thesecondary antibody and the conjugated oligonucleotides, a disulfide bondwas introduced. To achieve this, the antibody had to be modified priorto forming the disulfide bond with the oligonucleotides. In a firstattempt, the commercial reagent SPDP (cat #21857; Pierce) failed to givesatisfactory results. However, by following another approach andsynthesizing nitro-SPDP, we were able to modify the antibody.Mercaptopropionic acid in acetonitrile was treated with2,2′-dithiobis(5-nitropyridine) in the presence of triethylamine. Citricacid solution was added and the resulting 3-([5-nitro-2-pyridyl]dithio)propionic acid was extracted with dichloromethane. The product waspurified by silica gel flash chromatography. To prepare an active form,3-([5-nitro-2-pyridyl]dithio) propionic acid and N-hydroxysuccinimidewere dissolved in acetonitrile and N,N′-dicyclohexylcarbodiimide wasadded. Once the reaction was complete, crude nitro-SPDP was purified bypreparative thin layer chromatography (FIG. 26).

During antibody activation, nitro-SPDP precipitated out of solution andsubsequent successful activations were performed in the presence ofDMSO. Use of DMSO may not be suitable with primary antibodies.

The Nitro-SPDP activated antibody was subsequently desalted and mixedwith the A-METS and B-METS oligonucleotide at a 6-fold excess to performthe conjugation reaction involving the disulfide bond formation betweennitro-SPDP residues and thiol groups of the oligonucleotides. Thereaction efficiency was verified on a 10% native polyacrylamide gel(FIG. 27A) which was stained after electrophoresis with the nucleic acidstain GelRed to identify migration of DNA-protein complexes. Freeoligonucleotides will migrate quickly to the bottom of the gel, whileantibody-DNA complex migrating more slowly will be retained near the topof the gel. Detection of these slow migrating complexes with the DNAstain confirms the success of the conjugation reaction. The laddereffect observed where the antibody-DNA complexes migrate may be due tovarying DNA:antibody molar ratios, however this was not investigated.

Several purification and separation approaches were tested. However,both Protein A/G Spin Kit (cat #89980 from Pierce; manufacturer protocolwas used) and Thiophilic Adsorbtion Kit (cat #44916 from Pierce;manufacturer protocol was used) failed to achieve the desired separationof free oligonucleotide from antibody-oligo conjugate. Only sizeexclusion chromatography, using a HiLoad 16/600 Superdex 75 μg column,was able to purify the antibody-oligonucleotide conjugate and removefree oligos. The antibody-oligonucleotides reaction mixture was dilutedwith running buffer (50 mM Tris, 200 mM NaCl pH 8 and loaded onto theHiLoad 16/600 Superdex 75 μg column The running buffer was pumpedthrough the column at 1 ml/min till the chromatography was completed. UVabsorbance at three wavelengths was used as a detection method. FIG. 17illustrates the AKTA size exclusion chromatography profile at threewavelengths. The absorbance at 214 nm represents mostly proteinconcentration and the absorbance at 260 nm and 280 nm represent mostlyoligonucleotides concentration. Absorbance at 260 nm was used todetermine molar incorporation of oligonucleotide. Further, thepurification and separation was verified on a 10% native polyacrylamidegel by loading chromatography peaks 1 and 2 (FIG. 27B).

Functionality of Antibody-Oligonucleotide Conjugate in ChIP

A portion of the generated conjugate was tested by conventional ChIP toconfirm that the addition of the oligonucleotides did not impair primaryantibody binding. Chromatin was prepared as described above andApplicant's H3K4me3 antibody was used as the primary antibody with twodifferent ratios of secondary antibody-conjugate. Following enrichment,qPCR was performed using primers against untranslated region 12 (Untr12)and GAPDH. As illustrated in FIG. 28 columns 1 and 2, theantibody-conjugate still possesses binding activity and the optimalratio of primary H3K4me3 antibody to secondary antibody-conjugate is1:1. This experiment also contains the same data shown in FIG. 26 wherethe non-conjugated secondary was used to enable direct comparison(columns 3-7). Thus, while the function of the conjugate is attenuatedin ChIP (by three-fold), it was judged to be sufficiently robust topursue further testing.

Functionality of Antibody-DNA Conjugate in Transposomes

A series of experiments were performed to test whether oligonucleotidesnow tethered to an antibody would still associate with the transposometo form a functional transposome and to determine the optimal conditionsfor tagmentation with the antibody/transposome complex. First, since theantibody-conjugate was constructed using single-strandedoligonucleotides, the complementing p-MENTS sequence oligonucleotideswere added (FIG. 18), allowed to anneal for one hour at room temperature(1:1 molar ratio) to form double-stranded oligonucleotides. Thisconjugate was then incubated with the transposases at 1:1 molar ratiosfor one hour at room temperature to assemble the Tn5 transposomecomplex. The functionality of this conjugate was tested in ChIP toconfirm that the addition of oligonucleotides did not interfere with itsability to interact with rabbit primary antibodies. A two to three foldreduction was observed but deemed not significant enough to be ofconcern for this proof of concept stage of the project.

The ability of the antibody-tethered oligonucleotides to form afunctional transposome was also determined. Activity was first tested at55° C. for 30 min, the standard temperature for the Tn5 transposase, andalso for longer durations at 37° C. (FIG. 19B) due to concerns thatelevated temperatures when applied to the TAM-ChIP procedure woulddestabilize antibody-chromatin interactions. Tagmentation of purifiedgenomic DNA was detected at both temperatures. However, a portion ofuntagmented input DNA (arrow) remained in all conditions indicating thatthe tagmentation was incomplete. It was noted that less untagmented DNAremained in all TS-Tn5 reactions, suggesting that theTS-Tn5-transposome-antibody complex may be more robust than its Tn5counterpart. However, it is not clear if the incomplete tagmentationreactions with the transposome-conjugates are due to reduced transposaseactivity, incomplete or incorrect assembly of the transposome complex ordue to steric hindrance from being antibody-tethered. However, theresidual activity may nonetheless be sufficient for TAM-ChIP. Primaryantibody targeting of the transposome-antibody conjugate to chromatincould potentially overcome the decrease in random transposition activitythrough primary antibody mediated stabilization of thetransposome/antibody/chromatin complex, which would effectively drivethe reaction forward.

The assembled transposome was incubated with genomic MCF7 DNA at variousconcentrations ranging from 5 to 25 units and extending the incubationat 55° C. to 10 and 30 minutes. FIG. 29 shows that the tagmentationefficiency of the antibody/transposome complex is reduced. Untagementedinput DNA co-migrating with the band in the no enzyme (C− lanes)persists in reactions with the oligonucleotide-antibody conjugate. Thiscan be compared with reactions in FIG. 19 where no unfragmented DNA isdetectable in all enzyme containing reactions. While increasing theamount of enzyme did increase tagmentation, a longer incubation at 55°C. did not yield a significant improvement.

Since TAM-ChIP requires the antibody to still be bound to its targetduring the tagmentation reaction, transposase activation at 55° C. couldpotentially adversely affect antibody to remain bound to its targetprotein. Therefore, the tagmentation at a lower temperature wasassessed. Twenty-five units of the assembled Tn5 transposome wasincubated with genomic DNA at 37° C. and room temperature for varyingtimes to identify optimal conditions for tagmentation at lowertemperatures. In the initial experiment the three hour incubation at 37°C. resulted in a strong increase in tagmentation; however, when thisreaction was repeated, the same significant effect was not observed. Theinitial result was either an artifact of unequal sample loading on thegel (FIG. 30, compare lanes 3 and 4 with lanes 11 and 12), or thissample may have inadvertently received a higher amount of theantibody/transposome complex.

In the next experiment, twenty-five units of either the Tn5 and TS-Tn5transposome-conjugates were added to 1 μg of genomic MCF7 DNA andincubated at either 37° C. for one or three hours or 55° C. for one hour(FIG. 31, lanes marked C+). Interestingly, although both enzymes showreduced tagmentation activity when assembled to theoligonucleotide-conjugate, the TS-Tn5 transposome exhibited higheractivity than the Tn5 transposome with almost complete tagmentation at55° C. where unfragmented DNA is undetectable (compare lanes 2 and 6 inFIG. 31).

The reason for reduced activity of the antibody/transposome complex isnot clear. The incomplete tagmentation reactions with thetransposome-conjugates could be due to reduced transposase activity,incomplete or incorrect assembly of the transposome complex or due tosteric hindrance from being antibody-tethered. However, the residualactivity may nonetheless be sufficient for TAM-ChIP. Primary antibodytargeting of the transposome-antibody conjugate to chromatin couldpotentially overcome the decrease in random transposition activitythrough primary antibody mediated stabilization of thetransposome/antibody/chromatin complex, which would effectively drivethe reaction forward.

SUMMARY OF RESULTS

The data in this example demonstrate the development of a conjugationstrategy which was successful in the generation of an antibody-DNAconjugate. The data also demonstrate functionality of the conjugate inboth ChIP and transposase functions, albeit at attenuated levels. InChIP experiments, the same primary to secondary antibody ratiorequirement was retained post-conjugation. When assembled into atransposome, tagmentation of genomic DNA was demonstrable, indicatingthat the antibody-transposome complex was formed and functioned.

Example 13 TAM-ChIP Validation

To test the above hypothesis that antibody localization of transposaseto chromatin would overcome the attenuation of transposase activity thefollowing prototypic TAM-ChIP experiments were performed.

Functionality of Tn5 and TS-Tn5 Transposomes Assembled withAntibody-Oligonucleotide Conjugate in ChIP

A portion of the antibody-transposome complexes generated as describedwere used in a set of preliminary ChIP experiments. The H3K4me3 primaryantibody used above was incubated with 10 μg of chromatin overnight. Thetransposome-antibody conjugate, either Tn5 or TS-Tn5, was added at aratio of 1:1 of primary antibody to secondary-transposome conjugate, andincubated at 4° C. for four hours to allow binding of the secondaryantibody-transposome to the primary antibody. The reaction was dilutedfirst with four volumes of the buffer used in the immunoprecipitationstep of traditional ChIP and one volume of Mg²⁺ containing tagmentationbuffer to activate the transposase during a three hour incubation at 37°C. Antibody bound chromatin tagmented by the transposome was capturedusing Protein G agarose beads (Invitrogen) and eluted followingestablished ChIP procedures in the presence of TCEP(Tris(2-Carboxyethyl)phosphine) to reduce the disulfide bonding linkingoligonucleotide with antibody. After Proteinase K treatment and reversalof formaldehyde crosslinks, achieved with a two hour incubation at 80°C., half of the eluted DNA was subjected to 25 cycles of the four primerPCR reaction shown in FIG. 9 using Primer 1+2 and Adapters 1b and 2b(Table 3) as described above FIG. 22. The PCR amplification productswere purified using Agencourt AMPure XP magnetic beads and eluted in 30μl of elution buffer. DNA concentration was measured using a Nanodropspectrophotometer and samples were diluted to 2 ng/μl.

Following dilution, qPCR was performed on 10 ng of DNA using primersagainst the negative control regions Untr12 and Untr20 as well aspositive control regions for H3K4me3 GAPDH and Zc3h13. FIG. 21 showsquantitation of the qPCR products for these loci.

In the above procedure, protein G agarose beads were used in lieu of thestreptavidin-beads to avoid complications stemming frombiotin-streptavidin interactions. Thus the oligonucleotides designed andused in this example lacked a biotin moiety.

FIG. 32 shows that the TS-Tn5 but not the Tn5 transposome complexeffectively tagmented the regions bound by the primary antibody at achromatin concentration of 10 μg. The gene deserts and untranslatedregions on chromosome 12 and 20 were not detected, indicating notagmentation by the transposase at these loci. These data are consistentwith those in FIG. 31, where Tn5 transposome/antibody complex exhibitedlower tagmentation when compared to the TS-Tn5 transposome/antibodycomplex. The data in FIGS. 32 and 33 are expressed as enrichment overinput and not as binding events per 1,000 cells because four primer PCRwas performed prior to qPCR analysis thereby eliminating the possibleequation to cell equivalents. Note this scale is three order ofmagnitude smaller.

One possible interpretation of these results is that the observedenrichment (FIG. 32) was not a reflection of the actual amplification oftagmented target DNA during the four primer PCR reaction but rather anamplification of untagmented H3K4me3 enriched DNA carried over from whenprotein G beads were used to isolate antibody/chromatin complexes ChIP.To account for this possibility, the remaining half of the eluted DNAwas diluted to mimic the PCR reaction steps performed above, but notsubjected to the actual four primer PCR amplification, followed bypurification with Agencourt AMPure XP magnetic beads. The purified DNAwas eluted in 30 μl, diluted to 2 ng/μl and qPCR performed on 10 ng ofDNA using negative (Untr12) and positive (GAPDH) control primers. WhileFIG. 33 shows the result that would have been expected from atraditional ChIP using the H3K4me3 antibody, as in FIG. 28, theenrichment relative to input is at least three fold lower. Thisreduction was determined to be significant, especially when consideringthat the modest amount of GAPDH was detect in the Tn5 reaction in FIG.33 translated to essentially undetectable levels following four primerPCR in FIG. 32. Thus while untagmented DNA may contribute to the signalsdetected in FIG. 32, it is likely small and insignificant.

Thus, the results depicted in FIG. 32 clearly demonstrate the successfuland specific tagmentation of H3K4me3 antibody bound regions by theTS-Tn5 transposome while the reduced tagmentation activity of the Tn5transposome conjugate resulted in no or undetectable levels oftagmentation of the target regions and was not used further.

To confirm the results achieved above and further optimize TAM-ChIPperformance, several variables were next introduced. These includedvarying amounts of chromatin (10 and 1 μg), antibody-transposaseconcentrations (1:1 and 2:1 ratios of secondary/transposase conjugaterelative to primary antibody) and incubation times of theTS-Tn5/secondary complex (2 and 4 hours) with primary antibody:chromatincomplexes before transposase activation. Antibody-bound DNA was isolatedas described above followed by four primer PCR. qPCR of amplified DNAwas performed using primers against Untr12 and GAPDH. This experimentwas performed twice with inconsistent results indicating thatoptimization will be required to achieve reproducibility (FIG. 34).

Both experiments showed specific association of the H3K4me3 with theGAPDH region and not with Untr12, the condition which produced thehighest capture of a H3K4me3-associated genomic regions was with 10 μgchromatin in both, but for FIG. 34A, an incubation time of two hourswith 1:2 ratios of primary to secondary/transposome was best, while inthe experiment in FIG. 34B, an incubation time of four hours of thesecondary: transposase complex that was added at a ratio of 1:1 relativeto the primary antibody gave better results

The experiments preformed in FIG. 34 included no primary antibody(TS-Tn5 transposome/secondary complex only) and no secondary antibodycontrols to ensure that the observed enrichment was actual tagmentationby the transposase at regions bound by the primary antibody and not onlytagmentation of any open chromatin regions accessible to the enzyme, acontrol reaction with no primary antibody was performed. Samples lackingprimary antibody (FIG. 34B) show no enrichment thus confirming that theobserved tagmentation is taking place only at chromatin regions bound bythe primary antibody. The next step in the validation of the TAM-ChIPmethodology is a genome wide comparison of the libraries generated viathe method developed herein with the H3K4me3 antibody and those bytraditional ChIP-Seq methods with the same antibody. To that end thetagmented DNA generated in the experiments depicted in FIGS. 32 and 34were prepared for sequencing on an Illumina platform. To confirm thatthe size of the generated library was within the correct range twoindependent library preparations were run on a 1.5% agarose gel. In bothlibraries a majority of fragments in the population range between200-400 bp in length an ideal size for next generation sequencing.

Sequencing and 50-nt reads were generated on a HiSeq sequencer andaligned to the human genome (hg19). Only uniquely aligning reads werekept and duplicate reads were removed, resulting in 545,182 alignmentsfor the TAM-ChIP sample. A control standard ChIP-Seq data set(traditional ChIP-seq on MCF7 chromatin using the H3K4me3 antibody) wasdown-sampled to the same number of alignments and analyzed in parallel.Signal maps were generated and the fragment densities in 32-nt binsalong the genome was determined. The resulting histograms werevisualized in the Integrated Genome Browser (IGB). Peak calling wasperformed using SICER (Zang et al., Bioinformatics 25, 1952-1958, 2009)at a standard cutoff of E-value=1. SICER identified 10,897 peaks forTAM-ChIP and 12,526 peaks for traditional ChIP, which are withinexpectations for this histone mark if compared to numerous previousassays and 91.5% of the TAM-ChIP peaks overlapped with the traditionalChIP peaks (FIG. 15).

When annotated with genes, it was found that 83.6% of the TAM-ChIP peaksand 84.2% of the traditional ChIP peaks were located in promoters(defined as −7500 to +2500 relative to TSS). In conclusion, thecorrelation between the TAM-ChIP and traditional ChIP data is extremelyhigh. In both assay, highest signals were seen at the transcriptionalstart site (TSS), as shown in the promoter profile (−5000 to +5000)generated by seqMINER in FIG. 35, which is consistent with publishedresults (Ye et al., 2011). The typical seqMINERS promoter profiles forH3K4me3 ChIP-seq data show double peaks as can be seen for thetraditional ChIP-seq in pink. The profile for the generated TAM-ChIPlibrary on the other hand does not show the exact same pattern. Thesedata indicate that although the generated library was strikingly similar(91.5%) to the traditional ChIP additional optimization of the currentTAM-ChIP library generation is required to achieve the optimal TAM-ChIPprotocol for multi-analyte purposes.

These results indicate that the TS-Tn5 transposase can be directed in aspecific manner to chromatin via an antibody specific for a chromatinassociated protein—in this case, a post-translational histonemodification that is associated with transcriptionally active regions ofthe genome. Together the results presented herein clearly establishproof of concept.

Example 14 Enrich for DNA Methylated Genomic Regions UsingTranspososome-Antibody/Oligonucleotide Complex

Approach:

A recombinant tagged methyl-binding protein (in this case a His taggedMBD2 and/or MBD3 protein(s)) binds to methylated DNA and an anti-Hisantibody that has been conjugated with the same oligonucleotidescontaining the NGS adaptor and transposase sequences that bind to themethyl binding protein such that upon transposase activation theoligonucleotide sequence is integrated into region of DNA methylation.

Uniqueness Relative to “TAM-ChIP”:

-   -   1) Uses genomic DNA as a substrate as opposed to chromatin    -   2) Uses DNA binding proteins instead of a primary antibody    -   3) An antibody that recognizes the tag on a protein delivers the        oligonucleotide sequence to the DNA    -   4) Can be combined with other DNA modification marks, including        hydroxymethylcytosine, carboxylcytosine and formylcytosine in        addition to methylcytosine

Here data is present utilizing the antibody directed insertion ofbarcodes/sequences using the transposase to determine DNA methylationlevels in genomic DNA will allow this method to crossover into the fieldof DNA methylation, which is also analyzed at genome-wide levels andwill also facilitate an emerging trend of studies that examine howdistinct epigenetic regulatory mechanisms overlap or are co-integrated.

This approach enables the multianalyte capabilities of this assay as5-mC, 5-hmC, 5-caC and 5-fC and can be performed on unfragmented DNAwhich would likely reduce input DNA amounts compared to over methylationenrichment technologies (e.g. MeDIP). Using this approach the librarypreparation step will be eliminated and it is possible that theimmunoprecipitation step may also be removed.

The method described herein is based on Applicant's MethylCollector™assay, which enriches CpG-methylated DNA from limited amounts of cell ortissue samples (FIG. 37). The method is based on the Methylated CpGIsland Recovery Assay (MIRA), which utilizes the high affinity of theMBD2b/MBD3L1 complex for methylated DNA. In MethylCollector™ andMethylCollector™ Ultra His-tagged recombinant MBD2b, either alone(MethylCollector™) or in a complex with MBD3L (MethylCollector™ Ultra)to increase affinity, is incubated with fragmented DNA and itspecifically binds to CpG-methylated DNA. These protein-DNA complexesare then captured with nickel-coated magnetic beads and subsequent washsteps are performed to remove fragments with little or no methylation.The methylated DNA is then eluted from the beads in the presence ofProteinase K and enriched DNA can be used in many downstreamapplications, such as end point or real time PCR analysis, bisulfiteconversion, microarray analysis, sequencing, etc.

The first step to determine the feasibility of adapting the describedMethylCollector™ protocol for use with the transposome/antibodyconjugate was to confirm that MBD2b binding was retained in conditionsrequired for the tagmentation (i.e. low magnesium and 37°). The modifiedMethylCollector™ protocol included the necessary steps for binding ofthe anti-His antibody and tagmentation of the DNA by the transposome(FIG. 38).

The experiment was performed in duplicate with either 4 ug of MBD2balone or together with MBD3L on 100 ng of fragmented human genomic DNAfollowing either the original MethylCollector protocol (FIG. 37) or themodified protocol (FIG. 38). As depicted in FIG. 39, the modifiedprotocol (Test) shows some loss in sensitivity, but overall the resultswere promising as the methylated positive control region, NBR2, showsignificant enrichment over the unmethylated negative control region,GAPDH.

Based on these results an antibody conjugate directed against the Histagged MBD2 was developed. The transposase recognition sequences (A- andB-METS) was conjugated to an anti-His antibody (Pierce 6His epitope tagantibody (MA1-21315)) using the same conjugation strategy as describedpreviously. The retained activity of the conjugated antibody wasconfirmed by dotblot against 0-1000 ng of His-tagged recombinant MBD2b(data not shown).

After confirming the retained activity of the anti-His antibody, wefurther confirmed the functionality of the assembled conjugatedantibody-transposome complex. Unfragmented genomic DNA was incubatedwith 25 units of the assembled transposome complex and incubated at 50°for five minutes, or at 37° for either 1 or 3 hours. All reactionsshowed the same level of tagmentation and fragmentation of the genomicDNA compared to control reactions using an unconjugated transposomecomplex (data not shown)

The next step was to determine the feasibility of utilizing thisantibody conjugate in the MethylCollector™ assay and generating specifictagmentation by the transposome of only those regions containing DNAmethylation. Applicant incubated the His-tagged MBD2b and the anti-Hisantibody conjugate/transposome at a 1:1 ratio (4 ug of MBD2b and 4 ug ofthe anti-His antibody) with 100 ng unfragmented human genomic DNA (FIG.40). For half the samples the bound DNA was captured using the nickelcoated magnetic beads prior to purification (beads 1-3) and for theother half the capture step was omitted and all DNA purified (No Beads1-3). Each condition was performed in triplicate. The purified DNA wassubjected to 25 cycles of PCR amplification using the same adapters andprimers utilized in TAM-ChIP. All libraries were purified using standardmethods and diluted to 2 ng/ul. Quantitative PCR was performed usingprimers targeting the regions known to be negative (Untr12) or positive(GEMIN4) for DNA methylation.

FIG. 41 shows that the anti-His antibody conjugate-transposome complexeffectively tagmented those regions bound by the MBD2 protein, such asGEMIN4. Further, the untranslated region on chromosome 12 known to beunmethylated, showed no enrichment, indicating no tagmentation by thetransposase, confirming that the MBD2b protein was directing thetransposase to those DNA loci associated with MBD2b.

As one of the main advantages of the TAM-MIRA™ methodology (as disclosedherein) is its putative multianalyte capability, Applicant has initiatedstudies to show that this approach can be applied to determine 5-hmClevels in genomic unfragmented DNA. The advantages in studying 5-hmCcompared to 5-mC enrichment is that one can utilize an antibody such asthe Active Motif 5-Hydroxymethylcytosine antibody that recognizes andbinds both single- and double-stranded DNA. (An alternative approach isto use tagged hmc-binding proteins followed by transpososome complexescontaining an antibody to the tag).

Thus in this approach, Applicant incubated 100 ng of unfragmented mousegenomic DNA from brain and embryonic stem cells (ESC) with 2 ug of theprimary 5-hmC antibody followed by 2 ug of the secondary antibodyconjugate/transposome complex utilized in TAM-ChIP. After binding,dilution and tagmentation of the genomic DNA, the bound regions werecaptured using either Protein G Agarose or Magnetic beads, followed bywashes and elution. The purified DNA was subjected to 25 cycles of PCRamplification using the same adapters and primers utilized in TAM-ChIP.All libraries were purified using standard methods and diluted to 2ng/ul. Quantitative PCR was performed using primers targeting theregions known to be negative (Control 1) or positive (Slc22a4) for DNAhydroxymethylation. FIG. 42 demonstrates that the secondary antibodyconjugate/transposome complex can be utilized to specifically bind andtagment only those genomic loci bound by the 5-hmc antibody. Further,the negative control region known to be depleted of 5-hmC, showed noenrichment, indicating no tagmentation by the transposase, confirmingthat the 5-hmC antibody was directing the secondary antibody/transposometo those DNA loci associated with hydroxymethylation.

All together the results in this Example indicate that the methodologyof antibody directed tagmentation of unfragmented genomic DNA, inaddition to chromatin, by the TS-Tn5 transposase can be utilized tospecifically detect 5-mC and 5-hmC levels. These data also support thisapproach for detecting formyl and carboxylcytosine.

Those of skill in the art will recognize that many equivalentantibody/oligonucleotide conjugation strategies could be substituted foruse in the invention. For example, direct via a chemical crosslinker,indirect via other proteins/biomolecules that have strong interactions,including a streptavidin-protein A fusion protein (or protein G).Protein A binds the antibody in a manner that is known not to interferewith antibody function. A single protein A/G immunoglobulin bindingdomain could be also used, and expressed as a fusion protein. This wouldthen bind with biotinylated oligonucleotides. There are alsobiotin-binding peptides that are much smaller than the streptavidinprotein. Further, as indicated herein other transposon-targetedconstructs are possible including those described herein based onprotein-protein interactions, RNA-protein interactions, andDNA-DNA-interactions.

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We claim:
 1. A method of making a nucleic acid sequence library orlibraries comprising: a. extracting chromatin from a sample; b. addingat least one protein-oligonucleotide conjugate comprising an extractionmoiety to said chromatin; c. allowing said protein conjugates to locateat its/their target proteins and/or target DNA-binding sites and/ornoncoding RNA-binding sites in said chromatin d. tagging the nucleicacid in said chromatin fragments with said conjugate by inducing anintermolecular reaction between said oligonucleotide and said nucleicacid; and e. extracting the nucleic acids tagged using the extractionmoiety.
 2. A method according to claim 1 wherein theprotein-oligonucleotide conjugate comprises transposase and theintermolecular reaction is transposition.
 3. A method of making anucleic acid sequence library or libraries comprising: a. extracting andoptionally fragmenting chromatin from a sample; b. adding at least oneoligonucleotide-transposome construct comprising an extraction moiety tosaid chromatin; c. allowing said oligonucleotide-transposome constructto locate at its/their target DNA and/or noncoding RNA-binding sites insaid chromatin; d. tagging the nucleic acid in said chromatin fragmentswith said construct by inducing an intermolecular reaction between saidoligonucleotide and said nucleic acid; and e. extracting the nucleicacids tagged using the extraction moiety.
 4. A method according to claim3 wherein the oligonucleotide-transposome construct comprisestransposase and the intermolecular reaction is transposition.
 5. Theoligonucleotide of claim 3, wherein the oligonucleotide targetsnon-coding RNA.
 6. The oligonucleotide of claim 3, wherein theoligonucleotide contains peptide nucleic acid that targets G-quadruplexstructures.
 7. A method according to claim 1, wherein the extractionmoiety is a biotin molecule.
 8. A protein-transposome complex,comprising a protein that binds a protein-binding partner, methylatedDNA, non-coding RNA, or DNA-binding site.
 9. Anoligonucleotide-transposome construct, comprising an oligonucleotidethat hybridizes to non-coding RNA or G-quadruplex structures.
 10. Aprotein-transposome complex according to claim 8, wherein the protein isselected from the group consisting of one or more of bZIP domain,DNA-binding domain, helix-loop-helix, helix-turn-helix, MG-box, leucinezipper, lexitropsin, nucleic acid simulations, zinc finger, histonemethylases, recruitment proteins, Swi6 or chromodomain.
 11. Aprotein-transposome complex according to claim 8, wherein the protein isMBD2.
 12. A method according to claim 1, wherein the chromatin has beencrosslinked to proteins in vivo.
 13. A method according to claim 1,wherein the extracted tagged DNA is sequenced.
 14. A kit for practicingthe method according to claim 1, wherein said kit contains reagents,protein-transposome complex and/or oligonucleotide-transposomeconstruct, and instructions for use.
 15. The transposome of claim 8,wherein the transposon is Tn5 or TS-Tn5.
 16. (canceled)
 17. (canceled)18. A method according to claim 3, wherein the extraction moiety is abiotin molecule.
 19. A method according to claim 3, wherein thechromatin has been crosslinked to proteins in vivo.
 20. A methodaccording to claim 3, wherein the extracted tagged DNA is sequenced.